Assays for screening compounds which interact with cation channel proteins, mutant prokaryotic cation channel proteins, and uses thereof

ABSTRACT

Assays for screeing potential drugs or agents that can interact and potentially bind to cation channel proteins, and potentially have uses in treating conditions related to the function of cation channel proteins is provided, along with prokaryotic cation channel proteins mutated to mimic eukaryotic cation channels, which can then be used in assays of the present invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation in part of copending U.S. applicationSer. No. 09/054,347 filed on Apr. 2, 1998, which is a continuation inpart of copending U.S. application Ser. No. 09/045,529 filed on Mar. 20,1998, wherein both U.S. Ser. Nos. 09/054,347 and 09/045,529 are herebyincorporated by reference in their entireties.

GOVERNMENT RIGHTS CLAUSE

The research leading to the present invention was supported in part withNational Institutes of Health Grant GM 43949. The government may haverights in the invention.

FIELD OF INVENTION

The present invention relates to a crystal of a cation channel protein,and methods of using such a crystal in screening potential drugs andtherapeutic agents for use in treating conditions related to thefunction of such channels in vivo.

BACKGROUND OF INVENTION

Although numerous types of channel proteins are known, the main types ofion channel proteins are characterized by the method employed to open orclose the channel protein to either permit or prevent specific ions frompermeating the channel protein and crossing a lipid bilayer cellularmembrane. One important type of channel protein is the voltage-gatedchannel protein, which is opened or closed (gated) in response tochanges in electrical potential across the cell membrane. Another typeof ion channel protein are celled mechanically gated channel proteins,for which a mechanical stress on the protein opens or closes thechannel. Still another type is called a ligand-gated channel, whichopens or closes depending on whether a particular ligand is bound theprotein. The ligand can be either an extracellular moiety, such as aneurotransmitter, or an intracellular moiety, such as an ion ornucleotide.

Presently, over 100 types of ion channel proteins have been described,with additional ones being discovered. Basically, all ion channels havethe same basic structure regarding the permeation of their specific ion,although different gating mechanisms (as described above) can be used.One of the most common types of channel proteins, found in the membraneof almost all animal cells, permits the specific permeation of potassiumions (K⁺) across a cell membrane. In particular, potassium ions permeaterapidly across cell membranes through K⁺ channel proteins (up to 10⁸ions per second). Moreover, potassium channel proteins have the abilityto distinguish among potassium ions, and other small alkali metal ions,such as Li⁺ or Na⁺ with great fidelity. In particular, potassium ionsare at least ten thousand times more permeant than sodium ions. In lightof the fact that both potassium and sodium ions are generally sphericalin shape, with radii of about 1.33 Å and 0.95 Å respectively, suchselectivity is remarkable.

Broadly, potassium channel proteins comprise four (usually identical)subunits. Presently two major types of subunits are known. One type ofsubunit contains six long hydrophobic segments (presumablymembrane-spanning), while the other type contains two hydrophobicsegments. Regardless of what type of subunits are used, potassiumchannel proteins are highly selective for potassium ions, as explainedabove.

Among their many functions, potassium channel proteins control the paceof the heart, regulate the secretion of hormones such as insulin intothe blood stream, generate electrical impulses underlying informationtransfer in the nervous system, and control airway and vascular smoothmuscle tone. Thus, potassium channels participate in cellular controlprocesses that are abnormal, such as cardiac arrhythmia, diabetesmellitus, seizure disorder, asthma and hypertension, to name only a few.

Although potassium channel proteins are involved in such a wide varietyof homeostatic functions, few drugs or therapeutic agents are availablethat act on potassium channel proteins to treat abnormal processes. Areason for a lack of presently available drugs that act on potassiumchannel proteins is that isolated potassium channel proteins are notavailable in great abundance, mainly because an animal cell requiresonly a very limited number of such channel proteins in order tofunction. Consequently, it has been very difficult to isolate and purifypotassium channel proteins, reducing the amount of drug screeningefforts in search of potassium channel protein acting drugs.

Hence, what is needed is accurate information regarding the structure ofcation channel proteins so that drugs or therapeutic agents having anappropriate structure to potentially interact with a cation channelprotein can be selected.

What is also needed is an ability to overcome the physical limitationsregarding the isolation and purification of cation channel proteins,particularly potassium ion channel proteins.

What is also needed is a reliable method of utilizing cation channelproteins in screening potential drugs or agents for their possible usein treating conditions related to the function of cation channelproteins in vivo.

What is also needed are novel methods of using accurate informationregarding the structure of cation channel proteins so that drugs ortherapeutic agents can be screened for potential activity in treatingabnormal control processes of the body.

The citation of any reference herein should not be construed as anadmission that such reference is available as “Prior Art” to the instantapplication.

SUMMARY OF THE INVENTION

There is provided, in accordance with the present invention, a method ofpreparing a functional cation channel protein for use in an assay forscreening potential drugs or other agents which interact with a cationchannel protein, which permits the screening of potential drugs oragents that may be used as potential therapeutic agents in treatingconditions related to the function of cation channel proteins in vivo.

More specifically, the method comprising the steps of providing afunctional cation channel protein, conjugating the functional cationchannel protein to a solid phase resin, contacting the potential drug oragent to the functional cation channel protein conjugated to the solidphase resin, removing the functional cation channel protein from thesolid phase resin, and determining whether the potential drug or agentis bound to the cation channel protein.

In particular, the present invention extends to a method of preparing afunctional cation channel protein for use in an assay as describedabove, wherein the providing step of the method comprises expressing anisolated nucleic acid molecule encoding the cation channel protein in aunicellular host, such that the cation channel protein is present in thecell membrane of the unicellular host, lysing the unicellular host in asolubilizing solution so that the cation channel protein is solubilizedin the solution, and extracting the cation channel protein from thesolubilizing solution with a detergent. In a preferred embodiment, theisolated nucleic acid molecule comprises a DNA sequence of SEQ ID NO:17,or degenerate variants thereof, or an isolated nucleic acid moleculehybridizable under standard hybridization conditions to an isolatednucleic acid molecule having a DNA sequence of SEQ ID NO:17, ordegenerate variants thereof.

Numerous methods of lysing a unicellular host are known to the skilledartisan, and have applications in the present invention. In a preferredembodiment, lysing the unicellular host in a solubilizing solutioncomprises sonicating the unicellular host in a protein solubilizingsolution comprising 50 mM Tris buffer, 100 mM KCl, 10 mM MgSO₄, 25 mgDNAse 1, 250 mM sucrose, pepstatin, leupeptin, and PMSF, pH 7.5.

Furthermore, a skilled artisan is aware of numerous detergents that canbe used to extract an integral membrane bound protein, such as a cationchannel protein, from a solubilizing solution described above. Examplesof such detergents include SDS, Triton-100, Tween 20, Tween 80,glycerol, or decylmaltoside, to name only a few. Preferably, 40 mMdecylmaltoside is used to extract the cation channel protein from thesolubilizing solution.

Moreover, numerous solid phase resins to which a functional cationchannel protein can be conjugated have applications in a method ofpreparing a functional cation channel protein for use in an assay, asdescribed above. For example, a solid phase resin comprising insolublepolystyrene beads, PVDF, polyethylene glycol, or a cobalt resin, to nameonly a few have application in the present invention. Preferably, acation channel protein is conjugated to a cobalt resin at a protein toresin ratio that allows for saturation of the resin with the cationchannel protein. Moreover, after conjugation, the cobalt resin ispreferably used to line a column having a volume of about 1 ml.

After the cation channel protein is conjugated to a solid phase resin,it is contacted with a potential drug or agent, which is given anopportunity to bind to the cation channel protein.

Subsequently, the cation channel protein is removed from the solid phaseresin, and analyzed to determine whether the potential drug or agent isbound thereto. Numerous methods of removing the cation channel proteinfrom the solid phase resin are known to those of ordinary skill in theart. In a preferred embodiment, wherein the solid phase resin is acobalt resin, the removing step comprises contacting the cation channelprotein conjugated to the solid phase resin with an imidazole solution.This solution readily cleaves any bonds conjugating the cation channelprotein to the resin, so that the protein can removed from the resin,and collected for further analysis to determine whether the potentialdrug or agent is bound to the protein.

After the cation channel protein has been removed from the resin, itmust be examined to determine whether the potential drug or agent isbound thereto. If bound, the drug or agent may have uses involved inmodulation of the function of a cation channel protein in vivo,including uses as a therapeutic agent in treating conditions related tothe function of cation channel proteins. Numerous analytical methods arepresently available to the skilled artisan for determining whether thepotential ligand is bound to the cation channel protein. Examples ofsuch methods include molecular weight analysis with SDS-PAGE,immunoassays using an antibody to the drug or agent. HPLC, or massspectrometry.

Furthermore, the present invention extends to a method of using afunctional cation channel protein in an assay for screening potentialdrugs or agents which interact with the cation channel protein, whereinthe potential drug or agent is a member of a library of compounds, whichis contacted to the cation channel protein. Examples of libraries havingapplications in the present invention include, but are not limited to, amixture of compounds, or a combinatorial library of compounds.Furthermore, examples of combinatorial compounds having applications inthe present invention include, but are not limited to, a phage displaylibrary, or a synthetic peptide library, to name only a few.

In another embodiment, the present invention extends to a prokaryoticcation channel protein mutated to mimic a functional eukaryotic cationchannel protein. More specifically. Applicant has discovered that allcation channel proteins from all organisms have a conserved structure.Hence, placing mutations in a potassium channel from a prokaryoticorganism, for example, can permit the use of the prokaryotic cationchannel protein in screening assays for drugs that may interact withspecific eukaryotic cation channel proteins. For example, a prokaryoticpotassium channel protein can be mutated to mimic a cardiac potassiumchannel protein, a venous potassium channel protein, or a neuropotassium channel of a human, to name only a few.

Hence, pursuant to the present invention, a prokaryotic potassiumchannel protein, a prokaryotic sodium channel protein, or a prokaryoticcalcium channel protein can be mutated to mimic a eukaryotic cationchannel protein.

Examples of prokaryotic organisms from which a prokaryotic cationchannel protein can be taken and mutated to mimic a eukaryotic cationchannel protein include E. coli, Streptomyces lividans, Clostridiumacetobutylicum, or Staphylcoccus aureus, to name only a few.Furthermore, such prokaryotic cation channel proteins can comprise anamino acid sequence of SEQ ID Nos: 1, 2, 3, or 7, or conserved variantsthereof. In a preferred embodiment, the prokaryotic cation channelprotein mutated to mimic a eukaryotic cation channel protein, whereinthe prokaryotic cation channel protein is a potassium channel proteinfrom Streptomyces lividans.

Furthermore, pursuant to the present invention, a prokaryotic cationchannel protein can be mutated to mimic eukaryotic potassium channelprotein, a eukaryotic sodium channel protein, or a eukaryotic calciumchannel protein. Preferably, the eukaryotic cation channel protein isproduced endogenously in a eukaryotic organism, such as an insect or amammal, for example. More specifically, pursuant to the presentinvention, a prokaryotic cation channel protein is mutated to mimic aeukaryotic cation channel protein endogenously produced in a eukaryoticorganism selected from the group consisting of Drosophila melanogaster,Homo sapiens, C. elegans, Mus musculus, Arabidopsis thaliana, parameciumtetraaurelia or Rattus novegicus, or having an amino acid sequencecomprising SEQ ID Nos: 4, 5, 6, 8, 9, 10, 11, 12, 13, or 14, orconserved variants thereof.

In a preferred embodiment, the present invention extends to aprokaryotic cation channel protein mutated to mimic a functionaleukaryotic channel protein, wherein the prokaryotic cation channelprotein is a potassium channel protein from Streptomyces lividanscomprising an amino acid sequence of SEQ ID NO:1 or degenerate variantsthereof, and the eukaryotic cation channel is a potassium channelprotein comprising an amino acid sequence of SEQ ID NO:4 or conservedvariants thereof. As a result, the mutated prokaryotic channel proteincomprises an amino acid sequence of SEQ ID NO:16, or conserved variantsthereof, which is encoded by an isolated nucleic acid moleculecomprising a DNA sequence of SEQ ID NO:17, or degenerate variantsthereof.

In another embodiment, the present invention extends to a method ofusing a crystal of a cation channel protein, as described herein, in anassay system for screening drugs and other agents for their ability tomodulate the function of a cation channel protein, comprising the stepsof initially selecting a potential drug or agent by performing rationaldrug design with the three-dimensional structure determined for acrystal of the present invention, wherein the selecting is performed inconjunction with computer modeling. After potential drugs or agents havebeen selected, a cation channel protein is contacted with the potentialdrug or agent. If the drug or therapeutic agent has potential use formodulating the function of a cation channel protein, a change in thefunction of the cation channel after contact with the agent, relative tothe function of a similar cation channel protein not contacted with theagent, or the function of the same cation channel protein prior tocontact with the agent. Hence, the change in function is indicative ofthe ability of the drug or agent to modulate the function of a cationchannel protein.

Furthermore, the present invention extends to extends to a method ofusing a crystal of a cation channel protein as described herein, in anassay system for screening drugs and other agents for their ability tomodulate the function of a cation channel protein, wherein the crystalcomprises a Na⁺ channel protein, a K⁺ channel protein, or a Ca²⁺ channelprotein.

The present invention further extends to a method of using a crystal ofa cation channel protein in an assay for screening drugs or other agentsfor their ability to modulate the function of a cation channel protein,wherein the crystal of the cation channel protein comprises an aminoacid sequence of:

-   -   residues 23 to 119 of SEQ ID NO:1 (Streptomyces lividans);    -   residues 61 to 119 of SEQ ID NO:2 (E. coli);    -   residues 61 to 119 of SEQ ID NO:3 (Clostridium acetobutylicum);    -   residues 61 to 119 of SEQ ID NO:4 (Drosophila melanogaster);    -   residues 61 to 119 of SEQ ID NO:5 (Homo sapiens);    -   residues 61 to 119 of SEQ ID NO:6 (Homo sapiens);    -   residues 61 to 119 of SEQ ID NO:7 (Paramecium tetraaurelia);    -   residues 61 to 119 of SEQ ID NO:8 (C. elegans);    -   residues 61 to 119 of SEQ ID NO:9 (Mus musculus);    -   residues 61 to 119 of SEQ ID NO:10 (Homo sapiens);    -   residues 61 to 119 of SEQ ID NO:11 (Arabidopsis thaliana);    -   residues 61 to 119 of SEQ ID NO:12 (Homo sapiens);    -   residues 61 to 119 of SEQ ID NO:13 (Rattus novegicus); or    -   residues 61 to 119 of SEQ ID NO:14 (Homo sapiens);        or conserved variants thereof.

In a preferred embodiment of a method of using a crystal of a cationchannel protein in an assay for screening drugs or other agents fortheir ability to modulate the function of a cation channel protein, thecrystal comprises a potassium channel protein, comprising amino acidresidues 23 to 119 of SEQ ID NO:1, a space grouping of C2, and a unitcell of dimensions of a=128.8 Å, b=68.9 Å, c=112.0 Å, and β=124.6°.

Moreover, it is important to note that a drug's or agent's ability tomodulate the function of a cation channel protein includes, but is notlimited to, increasing or decreasing the cation channel protein'spermeability to the specific cation relative the permeability of thesame or a similar not contacted with the drug or agent, or the samecation channel protein prior to contact with the drug or agent.

In a further embodiment, the present invention extends to a method ofusing a crystal of a cation channel protein, as set forth herein, in anassay system for screening drugs and other agents for their ability totreat conditions related to the function of cation channel proteins invivo, and particularly in abnormal cellular control processes related tothe functioning of cation channel protein. Such a method comprises theinitial step of selecting a potential drug or other agent by performingrational drug design with the three-dimensional structure determined fora crystal of the invention, wherein the selecting is performed inconjunction with computer modeling. After potential drugs or therapeuticagents are selected, a cation channel protein is contacted with thepotential drug or agent. If an interaction of the potential drug orother agent with the cation channel is detected, it is indicative of thepotential use of the drug or agent to treat conditions related thefunction of cation channel proteins in vivo. Examples of such conditionsinclude, but are not limited to, cardiac arrhythmia, diabetes mellitus,seizure disorder, asthma or hypertension, to name only a few.

Furthermore, a crystal of a cation channel protein used in the methodfor screening drugs or agents for their ability to interact with acation channel comprises an Na⁺ channel protein. K⁺ channel protein, orCa²⁺ channel protein. Hence, the method of the present invention can beused to screen drugs or agents capable of treating conditions related tothe function of such channels.

Moreover, the present invention extends to a crystal used in the methodfor screening drugs or agents for their ability to interact with acation channel protein comprising an amino acid sequence of:

-   -   residues 23 to 119 of SEQ ID NO:1 (Streptomyces lividans);    -   residues 61 to 119 of SEQ ID NO:2 (E. coli);    -   residues 61 to 119 of SEQ ID NO:3 (Clostridium acetobutylicum);    -   residues 61 to 119 of SEQ ID NO:4 (Drosophila melanogaster);    -   residues 61 to 119 of SEQ ID NO:5 (Homo sapiens);    -   residues 61 to 119 of SEQ ID NO:6 (Homo sapiens);    -   residues 61 to 119 of SEQ ID NO:7 (Paramecium tetraaurelia);    -   residues 61 to 119 of SEQ ID NO:8 (C. elegans);    -   residues 61 to 119 of SEQ ID NO:9 (Mus musculus);    -   residues 61 to 119 of SEQ ID NO:10 (Homo sapiens);    -   residues 61 to 119 of SEQ ID NO:11 (Arabidopsis thaliana);    -   residues 61 to 119 of SEQ ID NO:12 (Homo sapiens);    -   residues 61 to 119 of SEQ ID NO:13 (Rattus novegicus); or    -   residues 61 to 119 of SEQ ID NO:14 (Homo sapiens),        or conserved variants thereof.

In a preferred embodiment, a crystal used in a method for screeningdrugs or agents for their ability to interact with a cation channel,comprises amino acid residues 23 to 119 of SEQ ID NO:1, has a spacegrouping of C2, and a unit cell of dimensions of a=128.8 Å, b=68.9 Å,c=112.0 Å, and β=124.6°.

In yet another embodiment, the present invention extends to a method ofusing a crystal of a cation channel protein described herein, in anassay system for screening drugs and other agents for their ability topermeate through a cation channel protein, comprising an initial step ofselecting a potential drug or other agent by performing rational drugdesign with the three-dimensional structure determined for the crystal,wherein the selecting of the potential drug or agent is performed inconjunction with computer modeling. After a potential drug or agent hasbeen selected, a cation channel protein can be prepared for use in theassay. For example, preparing the cation channel protein can includeisolating the cation channel protein from the membrane of a cell, andthen inserting the cation channel protein into a membrane having a firstand second side which is impermeable to the potential drug or agent. Asa result, the cation channel protein traverses the membrane, such thatthe extracellular portion of the cation channel protein is located onthe first side of the membrane, and the intracellular portion of thecation channel protein is located on the second side of the membrane.The extracellular portion of the cation channel membrane can then becontacted with the potential drug or agent. The presence of the drug oragent in the second side of the membrane is indicative of the drug's oragent's potential to permeate the cation channel protein, and the drugor agent is selected based on its ability to permeate the cation channelprotein.

In addition, a crystal used in a method for screening drugs or agentsfor their ability to permeate a cation channel can comprise a Na⁺channel protein, a K⁺ protein channel, or a Ca²⁺ protein channel.

Furthermore, the present invention extends to the use of a crystal in anassay system for screening drugs and other agents for their ability topermeate through a cation channel protein, wherein the crystal comprisesan amino acid sequence of:

-   -   residues 23 to 119 of SEQ ID NO:1 (Streptomyces lividans);    -   residues 61 to 119 of SEQ ID NO:2 (E. coli);    -   residues 61 to 119 of SEQ ID NO:3 (Clostridium acetobutylicum);    -   residues 61 to 119 of SEQ ID NO:4 (Drosophila melanogaster);    -   residues 61 to 119 of SEQ ID NO:5 (Homo sapiens);    -   residues 61 to 119 of SEQ ID NO:6 (Homo sapiens);    -   residues 61 to 119 of SEQ ID NO:7 (Paramecium tetraaurelia);    -   residues 61 to 119 of SEQ ID NO:8 (C. elegans);    -   residues 61 to 119 of SEQ ID NO:9 (Mus musculus);    -   residues 61 to 119 of SEQ ID NO:10 (Homo sapiens);    -   residues 61 to 119 of SEQ ID NO:11 (Arabidopsis thaliana);    -   residues 61 to 119 of SEQ ID NO:12 (Homo sapiens);    -   residues 61 to 119 of SEQ ID NO:13 (Rattus novegicus); or    -   residues 61 to 119 of SEQ ID NO:14 (Homo sapiens);        or conserved variants thereof.

In a preferred embodiment, the crystal used in an assay system of thepresent invention for screening drugs and other agents for their abilityto permeate through a cation channel protein comprises amino acidresidues 23 to 119 of SEQ ID NO:1, has a space grouping of C2, and aunit cell of dimensions of a=128.8 Å, b=68.9 Å, c=112.0 Å, and β=124.6°.

Naturally, the present invention extends to an isolated nucleic acidmolecule encoding a mutant K⁺ channel protein, comprising a DNA sequenceof SEQ ID NO:17, or degenerate variants thereof.

Furthermore, the present invention extends to an isolated nucleic acidmolecule hybridizable to an isolated nucleic acid molecule encoding amutant K⁺ channel protein under standard hybridization conditions.

Moreover, isolated nucleic acid molecules of the present invention, anddescribed above, can be detectably labeled. Examples of detectablelabels having applications in the present invention include, but are notlimited to, radioactive isotopes, compounds which fluoresce, or enzymes.

The present invention further extends to an isolated nucleic acidmolecule encoding a mutant K⁺ channel protein, or degenerate variantsthereof, comprising an amino acid sequence of SEQ ID NO:16, or conservedvariants thereof.

In addition, the present invention extends to an isolated nucleic acidmolecule encoding a polypeptide comprising an amino acid sequence of SEQID NO:16, or conserved variants thereof, wherein the isolated nucleicacid molecule is hybridizable under standard hybridization conditions toan isolated nucleic acid molecule encoding a K⁺ channel protein, ordegenerate variants thereof.

Furthermore, the present invention extends to a mutant cation channelprotein comprising an amino acid sequence of SEQ ID NO:16, or conservedvariants thereof.

In addition, the present invention extends to a cloning vectorcomprising an isolated nucleic acid molecule, or degenerate variantsthereof, which encodes a mutant cation channel protein of the presentinvention, or conserved variants thereof, and an origin of replication.The present invention also extends to a cloning vector comprising anorigin of replication and an isolated nucleic acid molecule hybridizableunder standard hybridization conditions to an isolated nucleic acidmolecule, or degenerate variants thereof, which encodes a mutant cationchannel protein of the present invention.

Examples of cloning vectors having applications in the present inventioninclude, but are not limited to, E. coli, bacteriophages, plasmids, andpUC plasmid derivatives. More specifically, examples of bacteriophages,plasmids, and pUC plasmid derivatives having applications hereincomprise lambda derivatives, pBR322 derivatives, and pGEX vectors, orpmal-c, pFLAG, respectively.

Naturally, the present invention extends to an expression vectorcomprising an isolated nucleic acid molecule comprising a DNA sequenceof SEQ ID NO:17, or degenerate variants thereof, operatively associatedwith a promoter. In another embodiment, an expression vector comprisesan isolated nucleic acid molecule hybridizable under standardhybridization conditions to an isolated nucleic acid comprising a DNAsequence of SEQ ID NO:17, or degenerate variants thereof, operativelyassociated with a promoter.

Examples of promoters having applications in expression vectors of thepresent invention comprise immediate early promoters of hCMV, earlypromoters of SV40, early promoters of adenovirus, early promoters ofvaccinia, early promoters of polyoma, late promoters of SV40, latepromoters of adenovirus, late promoters of vaccinia, late promoters ofpolyoma, the lac the trp system, the TAC system, the TRC system, themajor operator and promoter regions of phage lambda, control regions offd coat protein, 3-phosphoglycerate kinase promoter, acid phosphatasepromoter, or promoters of yeast α mating factor.

Furthermore, the present invention extends to a unicellular hosttransformed or transfected with an expression vector of the presentinvention. Such a unicellular host can be selected from the groupconsisting of E. coli, Pseudonomas, Bacillus, Strepomyces, yeast, CHO,R1.1, B-W, L-M, COS1, COS7, BSC1, BSC40, BMT10 and Sf9 cells.

Naturally, the present invention extends to a method of producing amutant cation channel protein, comprising the steps of culturing aunicellular host transformed or transfected with an expression vector ofthe present invention under conditions that provide for expression ofthe isolated nucleic acid molecule of the expression vector andrecovering the mutant cation channel protein from the unicellular host.Moreover, such a method can also be used wherein the expression vectorcomprises a an isolated nucleic acid molecule hybridizable understandard hybridization conditions to an isolated nucleic acid moleculecomprising a DNA sequence of SEQ ID NO:17, or degenerate variantsthereof, operatively associated with a promoter.

The present invention further extends to an antibody having a mutantcation channel protein of the present invention as an immunogen. Morespecifically, an antibody of the present invention can be a monoclonalantibody, a polyclonal antibody, or a chimeric antibody. Furthermore, anantibody of the present invention can be detectably labeled. Examples ofdetectable labels having applications in the present invention include,but are not limited to, an enzyme, a chemical which fluoresces, or aradioactive isotope.

Broadly, the present invention extends to a crystal of a cation channelprotein having a central pore, which is found natively in a lipidbilayer membrane of an animal cell, such that the central porecommunicates with extracellular matrix and cellular cytosol, wherein thecrystal effectively diffracts x-rays to a resolution of greater than 3.2angstroms.

Moreover, the present invention extends to a crystal of a cation channelprotein as described above, wherein the cation channel protein comprisesa first layer of aromatic amino acid residues positioned to extend intothe lipid bilayer membrane proximate to the interface an extracellularmatrix and lipid bilayer membrane, a second layer of aromatic amino acidresidues positioned to extend into the lipid bilayer membrane proximateto the interface of cellular cytosol and said lipid bilayer membrane, atetramer of four identical transmembrane subunits, and a central poreformed by the four identical transmembrane subunits.

Moreover, the present invention extends to a crystal of a cation channelprotein described above, wherein each transmembrane, subunit comprisesan inner transmembrane alpha-helix which has a kink therein, an outertransmembrane alpha-helix, and a pore alpha-helix, wherein each subunitis inserted into the tetramer of the cation channel protein so that theouter transmembrane helix of each subunit contacts the first and secondlayers of aromatic amino acid residues described above, and abuts thelipid bilayer membrane. Moreover, the inner transmembrane helix of eachsubunit abuts the central pore of the cation channel protein, contactsthe first and second layers of aromatic amino acid residues, is tiltedby about 25° with respect to the normal of the lipid bilayer membrane,and is packed against inner transmembrane alpha helices of othertransmembrane subunits at the second layer of aromatic amino acidresidues forming a bundle of helices at the second layer. The porealpha-helix of each subunit is located at the first layer of saidaromatic amino acid residues, and positioned between inner transmembranealpha-helices of adjacent subunits, and are directed, in an amino tocarboxyl sense, towards the center of the central pore.

Furthermore, the present invention extends to a crystal described above,comprising a cation channel protein having a central pore, whichcomprises a pore region located at the first layer of aromatic aminoacid residues, and connected to the inner and outer transmembranealpha-helices of said subunits. More particularly, the pore regioncomprises about 25-45 amino acid residues, a turret connected to thepore alpha-helix and the outer alpha-helix, wherein turret is located atthe interface of said extracellular matrix and the lipid bilayermembrane. The pore region further comprises an ion selectivity filterconnected to the pore alpha-helix and the inner transmembranealpha-helix of each subunit. The ion selectivity filter extends into thecentral pore of the cation channel protein, and comprises a signatureamino acid residue sequence having main chain atoms which create a stackof sequential oxygen atoms along the selectivity filter that extend intothe central pore, and amino acid residues having side chains thatinteract with the pore helix. It is the signature sequence which enablesa cation channel protein to discriminate among the cation intended topermeate the protein, and other cations, so that only the cationintended to permeate the channel protein is permitted to permeate.

The central pore further comprises a tunnel into the lipid bilayermembrane which communicates with the cellular cytosol, and a cavitylocated within the lipid bilayer membrane between the pore region andthe tunnel, and connected to the them, such that the central porecrosses the membrane.

Furthermore, the structure of all ion channel proteins share commonfeatures, which are set forth in the crystal of a cation channel proteindescribed above. Consequently, the present invention extends to acrystal of a cation channel protein having a central pore and structure,as described above, wherein the cation is selected from the groupconsisting of: Na³¹, K⁺, and Ca²⁻. Hence, the present invention extendsto crystals of potassium channel proteins, sodium channel proteins, andcalcium ion channels, to name only a few. In a preferred embodiment, thecrystal of a cation channel protein comprises a crystal of a potassiumion channel protein.

In addition, a crystal of a cation channel protein of a presentinvention comprises the amino acid sequence of any presently known, orsubsequently discovered cation protein channel. Consequently, thepresent invention extends to a crystal of a cation channel proteinhaving a central pore, which is found natively in a lipid bilayermembrane of an animal cell, such that the central pore communicates withextracellular matrix and cellular cytosol, wherein the crystal comprisesan amino acid sequence of:

-   -   residues 23 to 119 of SEQ ID NO:1 (Streptomyces lividans):    -   residues 61 to 119 of SEQ ID NO:2 (E. coli);    -   residues 61 to 119 of SEQ ID NO:3 (Clostridium acetobutylicum):    -   residues 61 to 119 of SEQ ID NO:4 (Drosophila melanogaster):    -   residues 61 to 119 of SEQ ID NO:5 (Homo sapiens):    -   residues 61 to 119 of SEQ ID NO:6 (Homo sapiens);    -   residues 61 to 119 of SEQ ID NO:7 (Paramecium tetraaurelia);    -   residues 61 to 119 of SEQ ID NO:8 (C. elegans);    -   residues 61 to 119 of SEQ ID NO:9 (Mus musculus);    -   residues 61 to 119 of SEQ ID NO:10 (Homo sapiens);    -   residues 61 to 119 of SEQ ID NO:11 (Arabidopsis thaliana);    -   residues 61 to 119 of SEQ ID NO:12 (Homo sapiens);    -   residues 61 to 119 of SEQ ID NO:13 (Rattus novegicus); or        residues 61 to 119 of SEQ ID NO:14 (Homo sapiens);        or conserved variants thereof.

In a preferred embodiment, a crystal of the present invention having acentral pore, which is found natively in a lipid bilayer membrane of ananimal cell, such that the central pore communicates with extracellularmatrix and cellular cytosol, comprises an amino sequence of amino acidresidues 23 to 119 of SEQ ID NO:1, has a space grouping of C2, a unitcell of dimensions of a=128.8 Å, b=68.9 Å, c=112.0 Å, and β=124.6°.

Furthermore, the present invention extends to a crystal of a cationchannel protein having a central pore, which is found natively in alipid bilayer membrane of an animal cell, such that the central porecommunicates with extracellular matrix and cellular cytosol, wherein thechannel protein comprises a signature sequence comprising:

-   -   Thr-Val-Gly-Tyr-Gly-Asp (SEQ ID NO:15).

In another embodiment, the present invention extends to a method forgrowing a crystal of a cation channel protein having a central pore,which is found natively in a lipid bilayer membrane of an animal cell,such that the central pore communicates with extracellular matrix andcellular cytosol, by sitting-drop vapor diffusion. Such a method of thepresent invention comprises the steps of providing the cation channelprotein, removing a predetermined number of carboxy terminal amino acidresidues from the cation channel protein to form a truncated cationchannel protein, dissolving the truncated cation channel protein in aprotein solubilizing solution, such that the concentration of dissolvedtruncated channel protein is about 5 to about 10 mg/ml, and mixing equalvolumes of protein solubilizing solution with reservoir mixture at 20°C. Preferably, the reservoir mixture comprises 200 mM CaCl₂, 100 mMHepes, 48% PEG 400, pH 7.5, and the protein solution comprises (150 mMKCl, 50 mM Tris, 2 mM DTT, pH 7.5).

Moreover, the present invention extends to a method of growing a crystalof a cation channel protein as described above, wherein a crystal can begrown comprising any kind of cation channel protein. In particular, thepresent invention can be used to grow crystals of potassium channelproteins, sodium channel proteins, or calcium channel proteins, to nameonly a few.

Furthermore, the present invention extends to a method of growing acrystal of a cation channel protein, as described herein, wherein thecrystal comprises an amino acid sequence of:

-   -   residues 23 to 119 of SEQ ID NO:1 (Streptomyces lividans);    -   residues 61 to 119 of SEQ ID NO:2 (E. coli);    -   residues 61 to 119 of SEQ ID NO:3 (Clostridium acetobutylicum);    -   residues 61 to 119 of SEQ ID NO:4 (Drosophila melanogaster);    -   residues 61 to 119 of SEQ ID NO:5 (Homo sapiens);    -   residues 61 to 119 of SEQ ID NO:6 (Homo sapiens);    -   residues 61 to 119 of SEQ ID NO:7 (Paramecium tetraaurelia);    -   residues 61 to 119 of SEQ ID NO:8 (C. elegans);    -   residues 61 to 119 of SEQ ID NO:9 (Mus musculus);    -   residues 61 to 119 of SEQ ID NO:10 (Homo sapiens);    -   residues 61 to 119 of SEQ ID NO:11 (Arabidopsis thaliana);    -   residues 61 to 119 of SEQ ID NO:12 (Homo sapiens);    -   residues 61 to 119 of SEQ ID NO:13 (Rattus novegicus); or    -   residues 61 to 119 of SEQ ID NO:14 (Homo sapiens);        or conserved variants thereof.

Numerous methods can be used to provide a cation channel protein, foruse in growing a crystal. For example, traditional purificationtechniques such as gel filtration, HPLC, or immunoprecipitation can beused to purify cation channel proteins from the membranes of numerouscells. In another method, recombinant DNA technology can be used,wherein a nucleic acid molecule encoding the particular cation channelprotein can be inserted into an expression vector, which is then used totransfect a unicellular host. After transfection, the host can beinduced to express the nucleic acid molecule, and the particular cationchannel protein can be harvested from the membrane of the unicellularhost.

Moreover, numerous methods are available for removing a predeterminednumber of carboxy terminal amino acid residues from the cation channelprotein to form a truncated cation channel protein. For example,chemical techniques can be used to cleave a peptide bond between twoparticular amino acid residues in the carboxy terminus of the cationchannel protein. In another embodiment, the cation channel protein canbe contacted with a proteolytic enzyme, so that the predetermined numberof residues from the carboxy terminus are enzymatically removed from thecarboxy terminus of the cation channel protein, forming a truncatedcation channel protein. In a preferred embodiment, the cation channelprotein comprises a potassium channel protein having an amino acidsequence of SEQ ID NO:1, which is contacted with chymotripsin so thatresidues 1-22 are removed, forming a truncated potassium channel proteincomprising an amino acid sequence of residues 23-119 of SEQ ID NO:1.

This invention further provides for a prescreening method foridentifying potential modulators of potassium ion channel functioncomprising the steps of: (i) binding a soluble potassium ion channelprotein to a solid support where the ion channel has the scaffold of atwo-transmembrane-domain-type potassium ion channel and has a tetramericconfirmation; (ii) contacting the soluble potassium ion channel proteinof step i with a compound in an aqueous solution; and, (iii) determiningthe binding of the compound to the soluble potassium ion channelprotein.

In addition, this invention provides for a method of screening forcompounds which selectively bind to a potassium ion channel proteincomprising: (i) complexing a functional two-transmembrane-domain-typepotassium ion channel protein to a solid support; (ii) contacting thecomplexed protein/solid support with an aqueous solution said solutioncontaining a compound that is being screened for the ability toselectively bind to the ion channel protein; and, (iii) determiningwhether the compound selectively binds to the ion channel protein withthe provisoes that the potassium ion channel protein is in the form of atetrameric protein; and, when the protein is mutated to correspond tothe agitoxin2 docking site of a Shaker K⁺ channel protein bysubstituting amino acid residues permitting the mutated protein to bindagitoxin2, the protein will bind agitoxin2 while bound to the solidsupport, said substituting of residues being within the 36 amino aciddomain defined by −25 to +5 of the selectivity filter where the 0residue is either the phenylalanine or the tyrosine of the filter'ssignature sequence selected from the group consisting ofglycine-phenylalanine-glycine or glycine-tyrosine-glycine.

In a particular embodiment of the method for screening for compounds asdescribed, above, a prokaryote two-transmembrane-domain-type ion channelprotein is used, such as from Steptomyces lividans especially, the KcsAchannel. The channels can be either wild-type or mutated from awild-type protein. One mutation is confined to the 36 amino acid domaindefined by −25 to +5 of the selectivity filter where the 0 residue iseither the phenylalanine or the tyrosine of the filter's signaturesequence selected from the group consisting ofglycine-phenylalanine-glycine or glycine-tyrosine-glycine. The method ofthis invention includes the use of channel mutations where the proteinalteration involves the deletion of a subsequence of the native aminoacid sequence and replacement of that native sequence with a subseqeuncefrom the corresponding domain of a second and different ion channelprotein. The second ion channel protein can be from either a prokaryoteor an eukaryote cell.

The methods described above may be conducted using an aqueous solutioncomprises a nonionic detergent.

In addition to the methods of this invention, the invention furthercomprises a column having the channel proteins of this invention boundthereto. The proteins are as described herein.

The invention also provides for a non-natural and functionaltwo-transmembrane-domain-type potassium ion channel protein wherein thenon-natural protein is mutated in its amino acid sequence from acorresponding natural protein whereby the mutation does not prevent thenon-natural protein from binding agitoxin2 when the non-natural proteinis further mutated to correspond to the agitoxin2 docking site of aShaker K⁺ channel protein said docking site created by substitutingamino acid residues selected from within the 36 amino acid domaindefined by −25 to +5 of the Shaker K⁺ selectivity filter where the 0residue is either the phenylalanine or the tyrosine of the filter'ssignature sequence selected from the group consisting ofglycine-phenylalanine-glycine or glycine-tyrosine-glycine. It ispreferred that the non-natural protein so modified will binds to achannel blocking protein toxin with at least a 10 fold increase inaffinity over the native ion channel. The non-natural proteins includethose mutations described above for use on a solid support to identifymodulators of potassium ion function.

The invention further provides for a means to assess the adequacy of thestructural conformation of a two-transmembrane-domain-type potassium ionchannel protein for high through put assays comprising the steps of: (i)complexing a two-transmembrane-domain-type potassium ion channel proteinhaving a tetrameric form to a non-lipid solid support under aqueousconditions; (ii) contacting the complexed two-transmembrane-domain-typepotassium ion channel protein with a substance known to bind to thetwo-transmembrane-domain-type potassium ion channel protein when boundto lipid membrane wherein the substance also modulates potassium ionflow in that channel protein; and, (iii) detecting the binding of thesubstance to the complexed two-transmembrane-domain-type potassium ionchannel protein. The channel proteins can be wildtype proteins ormodified as described above. Optionally the contacting is done in thepresence of a non-ionic detergent and the substance for binding iseither a channel blocker or other modulator including a toxin.

What's more, the present invention extends to columns havingapplications in the methods of the invention. In particular, the presentinvention extends to a column comprising a solid support having boundthereto an ion channel having the scaffold of atwo-transmembrane-domain-type potassium ion channel and having atetrameric confirmation.

Furthermore, the present invention extends to a column as describedabove, wherein the ion channel is a non-natural and functionaltwo-transmembrane-domain-type potassium ion channel protein wherein thenon-natural protein is mutated in its amino acid sequence from acorresponding natural protein. Such a mutation does not prevent thenon-natural protein from binding a toxin, such as agitoxin2 when thenon-natural protein is further mutated to correspond to the agitoxin2docking site of a Shaker K⁺ channel protein. Numerous means areavailable to the skilled artisan to create the docking. A particularmeans to create the docking site comprises substituting amino acidresidues selected from within the 36 amino acid domain defined by −25 to+5 of the Shaker K⁺ selectivity filter where the 0 residue is either thephenylalanine or the tyrosine of the filter's signature sequenceselected from the group consisting of glycine-phenylalanine-glycine orglycine-tyrosine-glycine.

Accordingly, it is a principal object of the present invention toprovide a crystal comprising a cation channel protein.

It is another object of the present invention to provide a method forgrowing a crystal comprising a cation channel protein.

It is yet another object of the present invention to utilize informationon the structure of a cation channel protein obtained from a crystal ofthe present invention, in an assay system for screening potential drugsor agents that may interact with a cation channel protein. Interactionof the potential drug or agent with a cation channel protein includesbinding to a cation channel protein, or modulating the function of acation channel protein, wherein modulation involves increasing thefunction of a cation channel protein to allow more specific cations tocross a cell membrane, or decrease the function of a cation channelprotein to limit or prevent specific cations from permeating through theprotein and crossing the cell membrane. Such drugs or therapeutic agentsmay have broad applications in treating a variety of abnormalconditions, such as cardiac arrhythmia, diabetes mellitus, seizuredisorder, asthma or hypertension, to name only a few.

It is vet another object of the present invention to provide mutant formof a cation channel protein, preferably a potassium channel protein fromStreptomyces lividans, which binds to Agitoxin2, a toxin found inscorpion venom, in a manner very similar to that in which eukaryoticpotassium channel proteins bind to Agitoxin2. Consequently, a mutantcation channel protein of the present invention mimics a functionaleukaryotic potassium channel protein, and can serve as a model thereforin screening potential drugs or agents that may interact with aeukaryotic potassium channel protein.

It is still yet another object of the present invention to provide amethod of preparing functional cation channel proteins for use in screensystems for assaying potential drugs or therapeutic agents which mayhave applications in treating conditions related to the function ofcation channel proteins in vivo.

It is yet another object of the present invention to provide mutatedprokaryotic cation channel proteins which mimic eukaryotic cationchannel proteins. With these mutated prokaryotic cation channelproteins, drugs or other can be screened for potential interaction withcation channel proteins in vivo, and hence, potential use as therapeuticagents in treating conditions related to the function of cation channelproteins in vivo, such as cardiac arrhythmia, diabetes mellitus, seizuredisorder, asthma or hypertension, to name only a few.

These and other aspects of the present invention will be betterappreciated by reference to the following drawings and DetailedDescription.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. (A) Sequence alignment of selected K⁺ channels and cyclicnucleotide-gated channels. The numbering and secondary structuralelements for the Streptomyces lividans K⁺; channel (kcsa) is given abovethe sequences. Selectivity filter, red; lining of the cavity and innerpore, blue: residues in which the nature of the side chain is preserved(>50% similarity), grey. The sequences are: kcsa, Streptomyces lividansaccession number (acc) 2127577 (SEQ ID NO:1): kch, Escherichia coli acc902457 (SEQ ID NO:2); clost. Clostridium acetoburylicum (GenomeTherapeutics Corp.) (SEQ ID NO:3); Shaker, Drosophila melanogaster acc85110 (SEQ ID NO:4); hKv1.1, Homo sapiens acc 1168947 (SEQ ID NO:5);hDRK, Homo sapiens acc 345875 (SEQ ID NO:6); Parame, Parameciumtetraaurelia acc 643475 (SEQ ID NO:7); Caenorhabiditis elegans acc2218158 (SEQ ID NO:8); mSlo, Mus musculus acc 539800 (SEQ ID NO:9);cal_act, Homo sapiens acc 2832249 (SEQ ID NO:10); AKT1, Arabidopsisthaliana acc 2129673 (SEQ ID NO:11); herg. Homo sapiens acc 2135973 (SEQID NO:12): romk, Rattus norvegicus acc 547736 (SEQ ID NO:13): hgirk.Homo sapiens acc 1042217 (SEQ ID NO:14): olCNG, Homo sapiens acc 2493743(SEQ ID NO:18): rodCNG, Homo sapiens acc 539557 (SEQ ID NO:19). The lasttwo sequences, separate from the rest, are from cyclic nucleotide-gatedchannels, which are not K⁺ selective.

FIG. 2. Experimental electron density map. Stereo views of theexperimental electron-density map contoured at 1σ covering nearly anentire subunit (removed from the tetramer) of the final model. The mapwas calculated at 3.2 Å resolution with the following Fouriercoefficients: native-sharpened amplitudes and MIR solvent flattenedaveraged phases. (A) Foreground: map showing inner helix, loopstructures and selectivity filter; background: the pore helix and outerhelix. CPK spheres show positions of mercury atoms used as residuemarkers (from the top, marked residues are Leu86. Leu90 and Val93). (B)Alternative view, Foreground: pore helix and part of outer helix;background: selectivity filter and turret. CPK sphere marks position ofAla42. (C) Close up view of electron density.

FIG. 3. Views of the tetramer. (A) Stereo view of ribbon representationillustrating the three-dimensional fold of the kcsa tetramer viewed fromthe extracellular side. The four subunits are distinguished by color.(B) Stereo view from another perspective, perpendicular to that in (A).(C) Ribbon representation of the tetramer as an integral-membraneprotein. Aromatic amino acids present on the membrane-facing surface aredisplayed in black. (D) Inverted tepee architecture of the tetramer.These diagrams were prepared with MOLSCRIPT and RASTER-3D (33 of ExampleI).

FIG. 4. Mutagenesis studies on Shaker: Mapping onto the kcsa structure.Mutations in the voltage-gated Shaker K⁺ channel that affect functionare mapped to the equivalent positions in kcsa based on the sequencealignment. Two subunits of kcsa are shown. Mutation of any of the whiteside chains significantly alters the affinity of agitoxin2 orcharybdotoxin for the Shaker K⁺ channel (12 of Example I). Changing theyellow side chain affects both agitoxin2 and tetraethylammonium ion(TEA) binding from the extracellular solution (14 of Example I). Thisresidue is the external TEA site. The mustard-colored side chain at thebase of the selectivity filter affects TEA binding from theintracellular solution (the internal TEA site (15 of Example I)). Theside chains colored green, when mutated to cysteine, are modified bycysteine-reactive agents whether or not the channel gate is open,whereas those colored pink react only when the channel is open (16 ofExample I). Finally, the residues colored red (GYG, main chain only) areabsolutely required for K⁺ selectivity (4 of Example I). This figure wasprepared with MOLSCRIPT and RASTER-3D.

FIG. 5. Molecular surface of kcsa and contour of the pore. (A) A cutawayStereo view displaying the solvent-accessible surface of the K⁺ channelcolored according to physical properties. Electrostatic potential wascalculated with the program GRASP, assuming an ionic strength equivalentto 150 mM KCl and dielectric constants of 2 and 80 for protein andsolvent, respectively. Side chains of lysine, arginine, glutamate andaspartate residues were assigned single positive or negative charges asappropriate, and the surface coloration varies smoothly from blue inareas of high positive charge through white to red in negatively chargedregions. The yellow areas of the surface are colored according to carbonatoms of the hydrophobic (or partly so) side chains of severalsemi-conserved residues in the inner vestibule (Thr75. Ile100, Phe103,Thr107, Ala108, Ala111, Val115). The green CPK spheres representpotassium ion positions in the conduction pathway. (B) Stereo view ofthe internal pore running the length of the ion channel. Within a stickmodel of the channel structure is a three dimensional representation ofthe minimum radial distance from the center of the channel pore to thenearest van der Waals protein contact.

The display was created with the program HOLE (34 of Example I).

FIG. 6. Identification of permeant ion positions in the pore. (A) A Rb⁻difference Fourier map calculated to 4.0 Å and contoured at 6σ identifytwo strong peaks corresponding to ions in the selectivity filter (innerand outer ions) and a weaker peak corresponding to ions in the cavity(cavity ion). The inner ion density has two closely-spaced peaks. (B) ACs⁺ difference Fourier map calculated to 5.0 Å and contoured at 6σ showsthe inner and outer ion peaks in the selectivity filter. Both differenceFourier maps were calculated with Fourier coefficients:F(soak)-F(native-unsharpened) and MIR phases. (C) Electron density mapcontoured at 1 (showing diffuse density at the cavity ion position. Thismap was calculated with the following Fourier coefficients: unsharpenednative amplitudes and MIR solvent flattened phases (no averaginginformation was included).

FIG. 7. Two mechanisms by which the K⁺ channel stabilizes a cation inthe middle of the membrane. First, a large aqueous cavity stabilizes anion (green) in the otherwise hydrophobic membrane interior. Second,oriented helices point their partial negative charge (carboxyl end, red)towards the cavity where a cation is located.

FIG. 8. Detailed views of the K⁺ channel selectivity filter. (A) Stereoview of the experimental electron-density (green) in the selectivityfilter. The map was calculated with native-sharpened amplitudes andMIR-solvent-flattened-averaged phases. The selectivity filter of threesubunits is shown as a stick representation with several signaturesequence residues labeled. The Rb⁺ difference map (yellow) is alsoshown. (B) Stereo view of the selectivity filter in a similarorientation to (A) with the chain closest to the viewer removed. Thethree chains represented are comprised of the signature sequence aminoacids Thr, Val, Gly, Tyr, Gly (SEQ ID NO:15) running from bottom to top,as labeled in single letter code. The Val and Tyr side chains aredirected away from the ion conduction pathway, which is lined by themain chain carbonyl oxygen atoms. Two K⁺ ions (green) are located atopposite ends of the selectivity filter, roughly 7.5 Å apart, with asingle water molecule (red) in between. The inner ion is depicted as inrapid equilibrium between adjacent coordination sites. The filter issurrounded by inner and pore helices (white). Although not shown, themodel accounts for hydrogen bonding of all amide nitrogen atoms in theselectivity filter except for that of Gly77. (C) A section of the modelperpendicular to the pore at the level of the selectivity filter andviewed from the cytoplasm. The view highlights the network of aromaticamino acids surrounding the selectivity filter. Tyrosine 78 from theselectivity filter (Y78) interacts through hydrogen bonding and van derWaals contacts with two Trp residues (W67. W68) from the pore helix.

FIG. 9. Sequence alignment of residues 51 to 86 of kcsa K⁺ (SEQ. IDNO:1) and Shaker K⁻ (SEQ. ID NO:4) channel pore regions. The numberingfor kcsa is given above the sequences. Structural elements are indicated(5 of Example 11). Asterisks mark several Shaker K⁺ channel amino acidlocations where mutations influence Agitoxin2 binding (4, 8, 9 ofExample II). Arrows mark the three kcsa K⁺ channel amino acids mutatedin this study. The sequences are: kcsa, Streptomyces lividans accessionnumber (acc) 2127577 and Shaker, Drosophila melanogaster acc 85110.

FIG. 10. Mass Spectra of scorpion toxins before and after channel columnpurification. MALDI-TOF mass spectra of venom before purification (A)and after elution from a cobalt column in the absence (B) and presence(C) of attached mutant kcsa K⁺ channel. The accuracy of the massmeasurements (±0.3 Da) permitted identification of most of the majorpeaks in the mass spectra searched from databases of known toxins of theLeiurus quinquestriatus hebraeus scorpion (D). The kcsa-bindingcomponent labeled * could not be assigned to a known scorpion toxin. Thecomponent labeled X (4193.0 Da) binds nonspecifically to the column andwas not identified. MALDI_MS was performed with the MALDI matrix4-hydroxy-α-cyano-cinnamic acid (16 of Example II).

FIG. 11. Binding affinity of wild type and mutant Agitoxin2 to themutant kcsa K⁺ channel. (A) Quantity of radiolabeled Agitoxin2 bound to0.3 μl of cobalt resin saturated with the mutant kcsa K⁺ channel isshown as a function of the radiolabeled Agitoxin2 concentration (17 ofExample II). Each point is the mean±SEM of 4 measurements, except forthe 0.03 μM and 1.5 μM concentrations which are the mean±range of meanof two measurements. The curve corresponds to equation BoundAgitoxin2=A*{1+K_(d)/[Agitoxin2]}⁻¹, with equilibrium dissociationconstant K_(d)=0.62 μM and resin capacity A=16 pMoles. (B) Remainingbound fraction of radiolabeled wild type toxin is graphed as a functionof the concentration of unlabeled wild type toxin or mutant toxins K27Aor N30A (17). Each point is mean±SEM of 4 measurements for wild typeAgitoxin2 (squares) or mean±range of mean of 2 measurements for K27A(circles) and N30A (triangles) Agitoxin2 mutants. The curves correspondto equation Remaining BoundFraction={1+Kdhot/[Thot]}*{1+(Kdhot/[Thot])*(1+[Tcold]/Kdcold)}⁻¹ withlabeled toxin concentration Thot=0.06 μM, wild type toxin Kdhot=0.62 μM,and competing toxin dissociation constant Kdcold=0.62 μM (wild type), 81μM(K27A), and 27 μM (N30A). (C) CPK model of Agitoxin2 viewing theinteraction surface. Side chains of functionally important amino acidsare shown in red (4 of Example II). This figure was prepared using theprogram GRASP (19 of Example II).

FIG. 12. Docking of Agitoxin2 onto the kcsa K⁺ channel. (A) Molecularsurface of the pore entryway of the kcsa K⁺ channel (left) and Agitoxin2(right). The colors indicate locations of interacting residues on thetoxin and channel surfaces as determined by thermodynamic mutant cycleanalysis of the Shaker K⁺ channel-Agitoxin2 interaction (4,8 of ExampleII). The three pore mutations of the kcsa K⁺ channel used in this study(Q58A. T61S, R64D) were introduced into the channel model using theprogram O (19 of Example II). Indicated residues on the channel surfacecorrespond to the positions of the Shaker K channel equivalent residues(See FIG. 9) which couple to the indicated Agitoxin2 residues. (B) Thepattern of colors in (A) suggests the docking orientation shown by themain worm representation of Agitoxin2 placed manually onto the poreentryway. The side chain colors match the colored patches in (A). Gly10is shown as a green band on the worm. The mutant cycle coupling betweenresidues at Shaker 425 (mutant kcsa 58) and residue 10 of Agitoxin2comes about through substitution of a bulky side chain residue at eitherposition (4, 7 of Example II). Pictures were made using the programGRASP (19 of Example II).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery of a crystal of a cationchannel protein, in particular a potassium channel protein fromStreptomyces lividans, and a method of forming such crystals. Moreover,the present invention is based on the recognition that, based on thestructure of the crystalline cation channel protein, potential drugs andtherapeutic agents which can bind to cation channel protein can bescreened for their use in treating conditions related to the function ofcation channel proteins, particularly potassium channel proteins, invivo.

Furthermore, the present invention is based upon the discovery thatcation channel proteins from prokaryotic organisms, such as a potassiumchannel protein from Streptomyces lividans, have much similarity andconservation with eukaryotic potassium channel proteins. In particular,a mutated prokaryotic potassium channel protein binds to a particularscorpion toxin in much the same way a eukaryotic potassium channelprotein binds to the same toxin.

For purposes of this Application, the term “positioned to extend intothe lipid bilayer membrane proximate to the interface . . . ” indicatesthat aromatic side groups of amino acid residues interject into thelipid bilayer membrane from about 0 Å to about 5 Å from the interface ofthe lipid bilayer with either the extracellular matrix of the cellularcytosol, i.e., the point at which the lipid bilayer membrane meetseither the extracellular matrix or the cellular cytosol.

Moreover, for purposes of this Application, the term “kink” indicatesthe inner transmembrane alpha-helix comprises a slight bend in itsstructure. Moreover, the angle of the tilt of the inner transmembranehelix “normal of the lipid bilayer” indicates the amount of tilt in theinner membrane helix relative to a line perpendicular to the lipidbilayer membrane at a point at which the inner transmembrane alpha-helixwould have intersected the lipid bilayer membrane, had the innertransmembrane alpha-helix extended thereto.

Moreover, for purposes of this Application the “specific ion” refers thethe ion species intended to permeate a particular cation channelprotein. For example, if the K⁺ is the specific ion for a potassiumchannel protein, Na⁺ is the specific ion for a sodium channel protein,and Ca²⁺ is the specific ion for calcium channel protein.

Furthermore, an α-helix in a protein is found when a stretch ofconsecutive residues all have a phi,psi angle pair of approximately −60°and −50°, corresponding to the allowed region of a Ramachandran plot(Branden, C. And Tooze, J. Introduction to Protein Structure, GarlandPublishing. Inc. New York and London, 1991 p. 12 (this reference isincorporated by reference herein in its entirety).

Moreover, the term “bundle” of α-helices, as used herein, refers to thepacking at least two α-helices closely together by intercalating sidechains of residues of the helices in the physically interact withAgitoxin2 and are primarily responsible for conferring the ability of achannel protein to bind to Agitoxin2.

As used herein, the term “functional” refers to a channel protein whichis in a tetrameric form and having a confirmation that is sufficientlyreflective of the native protein in its natural environment so that whena compound binds to the functional channel protein that same compoundwould also bind to that protein in its natural environment. The test fordetermining if a channel protein is functional is provided below andrelies upon the ability of the protein to bind Agitoxin2 whendeliberately mutated to bind the toxin.

“Non-natural” refers to a potassium ion channel protein that has beenmodified or altered from a corresponding wild type protein. Typicallythe protein is altered in its primary amino acid sequence but fusionsand chimera to the N and C terminus are included as well as addition ofnon-protein components to available reactive sites.

As used herein, “natural” refers to a potassium ion channel proteinwhich is found in nature. This is referred to as a wildtype.

The term “mutated” as used herein refers to a potassium ion channelprotein that has been altered by deletion, substitution of addition ofamino acids.

As used herein, the phrase “selectivity filter” refers to the domain ofchannel ion protein that is responsible for the ability of the proteinto exclude one or a group of ions and to allow other ions to pass.

As used herein, the phrase “signature sequence” refers to a sequence ofamino acids which define the protein as that protein or as belonging toa group or family of proteins. For specific proteins the signaturesequence may be very conserved and be a unique identifier. For signaturesequences that define a family, the sequence would be relativelyhypervariable but conserved across the family.

Also, as used herein, “solid supports” refer to any non-soluble matrixupon which the potassium ion channel proteins of this invention may beattached.

As used herein, the phrase “structural confomation” refers to a physicalrelationship between amino acids within a protein. It is a relativestate which alters with salt concentration, temperature and hydrophobicnature of the solvent being used. Structural confirmation is bestdefined by function.

The phrase “tetrameric protein” used herein refers to a protein havingquaternary structure comprising 4 subunits which may be the same ordifferent.

As used herein, the phrase “two-transmembrane-domain type potassium ionchannel protein” refers to potassium channel monomer having two regionsof hydrophobicity with sufficient length to form transmembrane segments.Between these two segments must be found the potassium channel signaturesequence. When using the tyrosine or phenylalanine residue of thesignature sequence as a zero reference point, the first transmembranesegment would begin within approximately −61 residues of the referencepoint and the second transmembrane would end within approximately +42amino acids of the reference point. To identify the two transmembranedomains one can construct a a Kyte-Dolittle hydropathy plot of the aminoacids.

As used herein, the phrase “wild-type” protein refers to a protein suchas a potassium ion channel protein which is presented with a primaryamino acid sequence that is found in nature.

Isolation of a Functional Cation Channel Protein for Use in Assays toScreen Potential Drugs and Therapeutic Agents

This method of the present invention overcomes limitations of usingcation channel proteins in the development of drugs or therapeuticagents to treat conditions related to the function of cation channelproteins, and particularly potassium cation channel proteins in vivo,such as cardiac arrhythmia, diabetes mellitus, seizure disorder, asthmaor hypertension, to name only a few.

In particular, since cells need very few potassium channels in order tofunction, it is difficult to isolate functional potassium channels ingreat quantities. Moreover, recombinant techniques to have a cellproduce excess potassium channel proteins has met with only limitedsuccess. As a result, very few drugs or agents are currently availablewhich act on potassium channel proteins.

However, Applicant has discovered a method to isolate cation channelproteins, particularly potassium cation channel proteins, which can thenbe used in efficient assays to screen potential drugs and agents forinteraction with such proteins. In particular, disclosed herein is amethod of using a functional cation channel protein in an assay forscreening for potential drugs or agents that may bind to a cationchannel protein comprising, wherein the assay comprises the steps ofproviding a functional cation channel protein, conjugating thefunctional cation channel protein to a solid phase resin, contacting thepotential drug or agent to the functional cation channel proteinconjugated to the solid phase resin, removing the functional cationchannel protein from the solid phase resin, and determining whether thepotential drug or agent is bound to the cation channel protein.

Since cation channel proteins are trans membrane bound proteins, careshould be taken in their isolation. In particular, to preventdenaturation and a loss of functional activity, they require ahydrophobic environment. In a preferred embodiment, a functional cationchannel protein is provided by expressing an isolated nucleic acidmolecule encoding the cation channel protein in a unicellular host suchthat the cation channel protein is present in the cell membrane of theunicellular host, lysing the unicellular host in a solubilizing solutionso that the cation channel protein is solubilized in the solution, andextracting the cation channel protein from the solubilizing solutionwith a detergent.

Many solubilizing solutions are presently known to one of ordinary skillin art, which can solubilize a cation channel protein, and prevent itsdenaturation or proteolytic digestion. All such solutions areencompassed by the present invention. In a preferred embodiment, thesolubilizing solution comprises Tris buffer, 100 mM KCl, 10 mM MgSO₄, 25mg DNAse 1, 250 mM sucrose, pepstatin, leupeptin, and PMSF at pH 7.5.

Moreover, many detergents are available to the skilled artisan forextracting solubilized cation channel protein from a solubilizingsolution of the present invention. Examples of detergents havingapplications herein include SDS, Triton 100, glycerol, decylmaltoside,Tween-20, or Tween-80, to name only a few. In a preferred embodiment, a40 mM decylmaltoside is used to extract the cation channel protein froma solubilizing solution of the present invention.

Furthermore, Applicant has discovered that cation channel proteins,particularly potassium cation channel proteins, can be conjugatedchemically to a solid phase resin. As a result, the channel proteins areimmobilized and readily available in assays for screening drugs oragents that may bind to a cation channel protein. In a preferredembodiment, a cation channel protein is conjugated to a cobalt resinthrough a carboxyl terminal hexahistidine tag.

In preferred embodiment, cation channel proteins are conjugated to acobalt resin at a protein to resin ratio that allows for saturation ofthe resin with the cation channel protein. As a result, numerous cationchannel proteins are immobilized and available for contact with apotential drug or therapeutic agent to be screened pursuant to thepresent invention.

Moreover, numerous screening methods are available and encompassed bythe present invention. For example, the resin with the cation channelconjugated thereto can be incubated in a solution comprising thepotential drug or therapeutic agent. In another embodiment, the resincan be used to line a column, to which the potential drug or agent isadded. Preferably, a potassium ion channel protein from Streptomyceslividans comprising an amino acid sequence of SEQ ID NO:1, or conservedvariants thereof, is mutated to mimic a eukaryotic potassium channel,such as a potassium channel protein of Drosophila melanogastercomprising an amino acid sequence of SEQ ID NO:4, or conserved variants.Consequently, the mutated potassium channel protein of Streptomyceslividans comprising an amino acid sequence of SEQ ID NO:16 is conjugatedto a cobalt resin, which is then used to line a 1 ml column. Acomposition comprising the potential drug or agent to be screened forinteraction with a eukaryotic cation channel protein is then poured intothe column, so that the potential drug or agent can contact the mutatedprokaryotic cation channel protein conjugated to the cobalt membrane.

After contact, the cation channel proteins are removed from the resin,and examined for interaction binding with the potential drug or agent.Numerous methods of cleaving a protein from a solid phase resin areavailable to the skilled artisan, and included in the present invention.In a preferred embodiment, the removing step comprises contacting thecation channel protein conjugated to the resin to an imidazole solution.The cation channel proteins can then be collected, and examined forinteraction, i.e. binding, with the potential drug or therapeutic agent.

Furthermore, determining whether the drug or therapeutic agent is boundto the cation channel protein can be done with numerous methods. Forexample, molecular weight determinations can be made with SDS-PAGEcomparing the molecular weight of the cation channel protein notcontacted with the drug, to the molecular weight of the cation channelprotein contacted with the drug. Furthermore, other analytical methods,such as HPLC, mass spectrometry, or spectrophotometry, to name only afew, can be used to determine whether the drug or agent is bound to acation channel protein previously conjugated to a solid phase resin.

Moreover, screening potential drugs or agents which may bind a cationchannel protein may be performed on an individual basis, i.e. onepotential drug or agent at a time, or the present invention can be usedto screen whole libraries of compounds at one time such as a mixture ofcompounds or a combinatorial library, for potential drugs or agentswhich potentially bind to a cation channel protein. For example,combinatorial libraries which can be screened with the present inventioninclude, but are not limited to, a phage display library, in whichnumerous proteins and polypeptides are being express simultaneously,libraries comprising synthetic peptides.

Two-Transmembrane-Domain Type Potassium Ion Channel Proteins

As set forth above, two-transmembrane type potassium ion channelproteins are well known and structurally constitute one of the classesof potassium channels. They are found in a wide variety of organisms,both prokaryotic and eukaryotic where they serve the purpose ofcontrolling the influx or efflux of potassium ions across cellmembranes. Potassium channels as a class are tetrameric membraneproteins characterized by multiple transmembrane segments and a poreregion through which potassium ions flow. These channels may behomotetrameric, that is, consisting of four identical monomers, orheterotetrameric, consisting of four monomers which are not necessarilyidentical. The individual monomers of the heterotetrameric forms areusually structurally related, and may or may not form a functionalpotassium channel when reconstituted as homotetramers of themselves. Thepore region contains a signature sequence consisting ofglycine-tyrosine-glycine or glycine-phenylalanine-glycine. Each monomerin the tetrameric structure contributes to the formation of the poreregion, and each subunit contains a signature sequence.

To identify a putative protein as a two-transmembrane potassium channelmonomer, a Kyte-Dolittle hydropathy plot of the amino acid may beconstructed, and it should demonstrate two regions of hydrophobicitywith sufficient length to form transmembrane segments. Between thesesegments must be found the potassium channel signature sequence. Whenusing the tyrosine or phenylalanine residue of the signature sequence asa zero reference point, the first transmembrane segment would beginwithin approximately −61 residues of the reference point and the secondtransmembrane would end within approximately +42 amino acids of thereference point.

Potassium channel monomer subunits may be obtained by a variety ofmethods, including cloning by nucleic acid hybridization, cloning byantibody selection of expressed proteins, and using the polymerase chainreaction (PCR) with homologous or degenerate primer sets. One of skillin the art would be able to readily obtain DNA sequence encoding suchpotassium channels given a known DNA sequence or an antibody against thechannel itself.

Examples of proteins which have been cloned and identified astwo-transmembrane potassium ion channels include IRK3 as described inKoyama H, et al. Molecular cloning, functional expression andlocalization of a novel inward rectifier potassium channel in the ratbrain. FEBS Lett 341:303-7 1994; IRK3 as described in Morishige et al.,Molecular cloning and functional expression of a novel brain-specificinward rectifier potassium channel. FEBS Lett 346: 251-6, 1994; UKATPreported in Inagaki et al. Cloning and functional characterization of anovel ATP-sensitive potassium channel ubiquitously expressed in rattissues, including pancreatic islets, pituitary, skeletal muscle, andheart. J Biol Chem 270:5691-4; and GIRK2 reported in Ferrer et al.,Pancreatic islet cells express a family of inwardly rectifying K⁺channel subunits which interact to form G-protein-activated channels. JBiol Chem 270:26086-91 1995.

Mutations of Two-Transmembrane-Domain Type Potassium Ion ChannelProteins

The present invention further extends to introducing Agitoxin2 dockingsites into two-transmembrane-domain type potassium ion channel protein.Any two transmembrane cation channel protein presently known, orsubsequently discovered, can routinely be modified to bind agitoxin2using the protocols described infra. As explained herein, scorpiontoxins, such as agitoxin2, bind to an ion channel by making contact withall four subunits where they come together to form the pore. Hence, suchtoxins will only bind to the channel if the subunits have been properlyassembled. As a result, the binding of a toxin, such as agitoxin2, to anon-natural two transmembrane cation channel protein can be used toconfirm the template channel integrity or function, i.e., to confirm thetwo-transmembrane cation channel protein has been properly modified tomimic a functional eukaryotic two-transmembrane cation channel protein.

The general method for creating an agitoxin (or related scorpion toxin)binding site on the template channel is now described. Particularexamples of pore region sequences (toxin binding sequences) of fourtwo-transmembrane cation channel proteins having applications in thepresent invention are described below: Shakeraeagsensffksipdafwwavvtmttvgygdmtpvgfwgk Romk1anhtpcveningltsaflfsletqvtigygfrcvteqcat Mjanesvilmtvegwdfftafytavvtistvgygdytpqtflgkls KcsAvlaerpgaqlitypralwwsvetattvgygdlypvtlwgr

Shaker is a six-membrane spanning K channel from Drosophilamelanogaster, ROMK1 is a two membrane-spanning K channel from rat renalouter medulla (kidney). Mjan is a two membrane-spanning K channel fromMethanococcus janschii, and KcsA is a two membrane-spanning K channelfrom the bacterium Streptomyces lividans.

As explained herein, cation channle proteins have a high degree ofsequence conservation, particularly in the region of the selectivityfilter. Hence, gyg sequence should be used as a reference to align thesequences. The underlined amino acids on the Shaker channel sequence areknown to be important for binding of agitoxin, as described infra. Inparticular, described herein is the mutating of several of theunderlined amino acids, using standard techniques. As a result of thesemutations, the KcsA K channel became sensitive to agitoxin binding.Similarly, other channels can be subjected to the same analysis.Therefore, using the teachings set forth infra. Mjan or Romk1 channelscan readily be modified by those of ordinary skill. Numerous techniquesare readily available to the skilled artisan to convert the appropriate(underlined) amino acids of the pore relations of the two-transmembranecation channel proteins described above to the amino acid residues foundin the corresponding position of the Shaker K channel. A particulartechnique which can be in this modification process is directedmutagenesis.

Also, the present invention involves introducing mutations into thetwo-transmembrane-domain type potassium ion channel protein which allowit to mimic other potassium ion channel proteins. In particular, thepresent invention contemplates the use of two-transmembrane proteins asa scaffold for studying or identifying modulators of potassium ionchannel function. The proteins can be modified in a variety of differentways to mimic or simulate properties of related potassium ion channelsincluding conferring properties found in six membrane domain type ionchannels. Accordingly, one can create channel proteins that have beenminimally altered from their corresponding wild type for convenience ofpurification, i.e. removing protease cleavage sites in noncriticaldomains, or attaching binding domains to facilitate chromatographicpurifications such as FLAG or polyHis. Because the overall structure ofpotassium ion proteins is conserved, modifications can be introducedthat can transfer properties of one channel protein to thetwo-transmembrane proteins that is being used as a scaffold. Among thesemodifications are venom docking sites as exemplified herein as well asbinding sites for modulators such as to the transmembrane domains andalterations to the ion filter region.

Recombinant genetics has a variety of techniques for introducing and fordetermining the domains and in many cases the specific amino acids whichare responsible for the physical properties of channel proteins. Inbrief, these methods consists of manipulating the amino acid sequence ofa protein in order to identify which part of the protein is involved inthe structure or function of the molecule and then transferring thatdomain and its properties to proteins that do not naturally have thatproperty. These methods have already been widely applied in the study ofion channels. The study of ion channels lends itself very well to suchmethods, because these proteins exist in a number of functional familieswithin which are numerous structurally related yet biophysically andpharmacologically distinct subfamily members. For example, thesuperfamily of potassium channels all share the pore signature sequencegly-tyr-gly or gly-phe-gly, and are tetrameric; subfamily monomers mayhave two transmembrane segments or 6 transmembrane segments, and may begated by membrane potential, intracellular calcium concentration,intracellular cyclic nucleotides, membrane deformation, and pH: they maybe inwardly rectifying, outwardly rectifying, or nonrectifying: andtheir activation and inactivation kinetics, and conductances may varytremendously.

As exemplified in this application, a number of scorpion and bee venom,toxins can bind with high affinity to one subfamily member while beinginactive on a closely related subfamily members. It is therefore notsurprising that amino acid sequence mutations which confer theproperties of one ion channel upon another are a tool which has beencommonly employed by ion channel researchers and this invention takesadvantage of this plieomorphic property in the super family of potassiumchannels.

Mutations may be introduced using a number of approaches, each with itsown particular strengths. Often a combination of these may be used togenerate a channel with altered properties. Examples of these approachesare deletions of amino acids, domain replacement of one channel withthat of a different channel (chimeras), replacement of amino acids withdifferent amino acid in a nontargeted or semi-targeted way (e.g.alanine-scanning mutagenesis) and replacement of targeted amino acidswith different amino acids (site-directed mutagenesis). Although eachmethod may be applied independently, oftentimes several or all of thesemay be employed to arrive at a mutant channel with the desiredcharacteristics. Examples of changed characteristics include channelgating, voltage response, rectification, ion preference, and the bindingof small organic molecules and peptides to the channel.

Mutagenesis is especially powerful when an ion channel with novel toxinor small organic molecule-binding characteristics is required. Usingthis approach, channels which do not show significant binding of aparticular toxin or small organic molecule may be engineered to bindstrongly to these molecules. Conversely, channels which strongly bind aparticular toxin or small organic molecule may be engineered to losethat property.

Examples of the use of the chimeric and site-directed approach are many.In Ishii, T. M., Maylie, J. and Adelman, J. P. (1997) J. Biol. Chem 272:23195-200, the authors were able to confer apamin sensitivity on achannel which did not possess this property. Similar studies have beenperformed on the Kv1.3 and Kv2.1 potassium channels by Gross et al.(1994), Neuron 13: 961-6. In their study, they transferred scorpiontoxin sensitivity from the highly sensitive Kv1.3 potassium channel tothe insensitive Kv2.1 potassium channel by transferring the stretch ofamino acids between transmembrane domains 5 and 6. Conversely,alanine-scanning mutagenesis was used by Hanner et al. (1998). J BiolChem 273: 16289-96, to impair charybdotoxin binding to the maxi-Kchannel, and direct point mutations were employed by Wang and Wang(1998), Proc Natl Acad Sci USA 95:2653-8, to remove batrachotoxinsensitivity from sodium channels.

Mutagenesis may also be employed to alter the biophysical properties ofion channels, in effect causing one channel to have characteristicssimilar to those of another. For example, voltage-gated potassiumchannels of the Shaker subfamily open in response to changes in membranepotential. Members of this subfamily of potassium channels have theintrinsic property of opening at different membrane potentials dependingon the particular family member, and have the characteristic of delayedrectification. Liman et al., (1991). Nature, 353:752-6, were able todemonstrate that mutations in the S4 voltage sensor domain of Shakerchanged the opening potential; by mutating several amino acid residuesin the S4 voltage sensor domain of Shaker, Miller and Aldrich (1996),Neuron. 16:853-8, were able to convert this channel from a delayedrectifier into a voltage-gated inward rectifier. Chimeric constructs mayuse related domains from different channel types. The rat CNG olfactorychannel is a member of the voltage-gated subfamily of potassiumchannels, but is itself voltage-independent and is not entirelyselective for potassium ions as compared with the eag channel. Tang andPapazian (1997), J Gen Physiol, 109:301-11, were able to convert thehuman eag potassium channel from a voltage sensitive to avoltage-independent channel by substituting the S3-S4 domain of the ratcyclic-nucleotide gated (CNG) olfactory channel.

It is therefore clear that mutagenesis may be readily used to confer thepharmacological and biophysical properties of one channel upon another,and that this methodology applies to not only potassium, but sodium andcalcium channels.

Determining if the two-transmembrane-domain type potassium ion channelprotein has maintained function using Agitoxin2 binding. Beyond theability of the channel proteins of this invention to pass ions under exvivo conditions or using liposomes, their functionality can measured bythe ability to be modified to accept or recognize agitoxin2. Toaccomplish this one follows the mutagenesis methods described above bothgenerically for mutation of any channel protein and for the introductionof an agitoxin2 docking site into any two transmembrane-type domainpotassium ion channel protein.

Once mutated, the proteins are tested by any number of binding assayformats including homogenous assays where both agitoxin2 and the channelprotein are free in solution and heterogeneous assay formats where oneof the binding members is bound to a solid support. Either member can belabelled using the labels described herein. The preferred method forassaying for agitoxin2-binding uses the cobalt resin and proceduresdescribed in Example II.

Binding the Two-Transmembrane-Domain Type Potassium Ion Channel Proteinto Solid Supports

The potassium channels of the invention can be bound to a variety ofsolid supports. Solid supports of this invention include membranes(e.g., nitrocellulose or nylon), a microtiter dish (e.g. PVC,polypropylene, or polystyrene), a test tube (glass or plastic), a dipstick (e.g., glass, PVC, polypropylene, polystyrene, latex and thelike), a microfuge tube, or a glass, silica, plastic, metallic orpolymer bead or other substrate such as paper. A preferred solid supportuses a cobalt or nickel column which binds with specificity to ahistadine tag engineered onto the channel proteins.

Adhesion of the channel proteins to the solid support can be direct(i.e. the protein contacts the solid support) or indirect (a particularcompound or compounds are bound to the support and the target proteinbinds to this compound rather than the solid support). One canimmobilize channel proteins either covalently (e.g., utilizing singlereactive thiol groups of cysteine residues (see, e.g., Colliuod et al.Bioconjugate Chem. 4:528-536 (1993)) or non-covalently but specifically(e.g., via immobilized antibodies (Schuhmann et al. Adv. Mater.3:388-391 (1991); Lu et al. Anal. Chem. 67:83-87 (1995), thebiotin/strepavidin system (Iwane et al. Biophys. Biochem. Res. Comm.230:76-80 (1997) or metal chelating Langmuir-Blodgett films (Ng et al.Langmuir 11:4048-55 (1995); Schmitt et al. Angew. Chem. Int. Ed. Engl.35:317-20 (1996); Frey et al. Proc. Natl. Acad. Sci. USA 93:493741(1996); Kubalek et al. J. Struct. Biol. 113:117-123 (1994)) andmetal-chelating self-assembled monolayers (Sigal et al. Anal. Chem.68:490497 (1996)) for binding of polyhistidine fusions.

Indirect binding can be achieved using a variety of linkers which arecommercially available. The reactive ends can be any of a variety offunctionalities including, but not limited to: amino reacting ends suchas N-hydroxysuccinimide (NHS) active esters, imidoesters, aldehydes,epoxides, sulfonyl halides, isocyanate, isothiocyanate, and nitroarylhalides; and thiol reacting ends such as pyridyl disulfides, maleimides,thiophthalimides, and active halogens. The heterobifunctionalcrosslinking reagents have two different reactive ends, e.g., anamino-reactive end and a thiol-reactive end, while homobifunctionalreagents have two similar reactive ends, e.g., bismaleimidohexane (BMH)which permits the cross-linking of sulfhydryl-containing compounds. Thespacer can be of varying length and be aliphatic or aromatic. Examplesof commercially available homobifunctional cross-linking reagentsinclude, but are not limited to, the imidoesters such as dimethyladipimidate dihydrochloride (DMA); dimethyl pimelimidate dihydrochloride(DMP); and dimethyl suberimidate dihydrochloride (DMS).

Heterobifunctional reagents include commercially available activehalogen-NHS active esters coupling agents such as N-succinimidylbromoacetate and

N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB) and thesulfosuccinimidyl derivatives such assulfosuccinimidyl(4-iodoacetyl)aminobenzoate (sulfo-SIAB) (Pierce).Another group of coupling agents is the heterobifunctional and thiolcleavable agents such as N-succinimidyl 3-(2-pyridyidithio)propionate(SPDP) (Pierce).

Antibodies are also available for binding channel proteins to a solidsupport. This can be done directly by binding channel protein specificantibodies to the column and allowing channel proteins to bind or it canbe done by creating chimeras constructed from the channel protein linkedto an appropriate immunoglobulin constant domain sequence, they aretermed immunoadhesins and they are known in the art. Immunoadhesinsreported in the literature include Gascoigne et al., Proc. Natl. Acad.Sci. USA 84,. 2936-2940 (1987), Capon et al. Nature 377, 525-531 (1989);and Traunecker et al., Nature 33, 68-70 (1989).

By manipulating the solid support and the mode of attachment of thetarget molecule to the support, it is possible to control theorientation of the target molecule. Thus, for example, where it isdesirable to attach a target molecule to a surface in a manner thatleaves the molecule tail free to interact with other molecules, a tag(e.g., FLAG, myc. GST, polyhis, etc.) may be added t) the targetmolecule at a particular position in the target sequence.

It is also possible to reconstitute of channels in lipid, membranes orliposomes. For example the following references teach how toreconstitute the channel proteins of this invention in membranes. Thevery channels of this invention, SliK, the K+ channel encoded by theStreptomyces KcsA gene, was expressed, purified, and reconstituted inliposomes. See, Heginbotham L et al. J Gen Physiol 1998 June;111(6):741-9 and in Cuello L G, et al., Biochemistry 1998 Mar. 10;37(10):3229-36. In Shin, J H et al. FEBS Lett 1997 Oct. 6;415(3):299-302 where the authors demonstrated that nitric oxide couldactivate a calcium-activated potassium channel from rat using the planarlipid bilayer technique. Santacruz-Toloza L et al. Biochemistry 1994Feb. 15; 33(6): 1295-9.

Assays

Once bound there are a variety of assay formats that can be used toscreen for modulators of the channel proteins. Various molecules thatinteract with a potassium channel can be identified by 1) attaching thepotassium channel (“the target”) to a solid support, 2) contacting asecond molecule with the support coated with the potassium channel, and3) detecting the binding of the second molecule to the potassiumchannel. Molecules that interact or bind with the target are theneluted, with or without the target, thereby isolating molecules thatinteract with the target.

For a general description of different formats for binding assays, seeBASIC AND CLINICAL IMMUNOLOGY, 7^(th) Ed. (D. Stiles and A. Terr,ed.)(1991): ENZYME IMMUNOASSAY. E. T. Maggio, ed., CRC Press, BocaRaton, Fla. (1980); and “Practice and Theory of Enzyme Immunoassays” inP. Tijssen, LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY,Elsevier Science Publishres. B. V. Amsterdam (1985), each of which isincorporated by reference.

In competitive binding assays, the test compound competes with a secondcompound for specific binding sites on a target molecule attached to thesolid support. Binding is determined by assessing the amount of secondcompound associated with the target molecule. The amount of secondcompound associated with the target molecule is inversely proportionalto the ability of a test compound to compete in the binding assay.

The amount of inhibition or stimulation of binding of a labeled targetby the test compound depends on the binding assay conditions and on theconcentrations of binding agent, labeled analyte and test compoundsused. Under specified assay conditions, a compound is said to be capableof inhibiting the binding of a second compound to a target compound tothe amount of bound second compound is decreased by 50% or preferably90% or more compared to a control sample.

Alternatively, various known or unknown compounds, including proteins,carbohydrates, and the like, can be assayed for their ability to bind tothe channels of this invention. In one embodiment, samples from varioustissues are contacted with the target to isolate molecules that interactwith the target. In another embodiment, small molecule libraries andhigh throughput screening methods are used to identify compounds thatbind to the target.

Labels for Use in Assays

The amount of binding of the second compound to a target channel proteincan be assessed by directly labeling the second compound with adetectable moiety, or by detecting the binding of a labeled ligand thatspecifically binds to the second compound. A wide variety of labels canbe used. The detectable labels of the invention can be primary labels(where the label comprises an element that is detected or that producesa directly detectable signal) or secondary labels (where the detectedlabel binds to a primary label, e.g., as is common in immunologicallabeling). An introduction to labels, labeling procedures and detectionof labels is found in Polak and Van Noorden (1997) Introduction toImmunochemistry, 2^(nd) ed., Springer Verlag, N.Y. and in Haugland(1996) Handbook of Fluorescent Probes and Research Chemicals, a combinedcatalog and handbook published by Molecular Probes. Inc., Eugene, Oreg.Useful primary and secondary labels of the present invention can includespectral labels such as fluorescein isothiocyanate (FITC) and OregonGreen™, rhodamine and derivatives (e.g. Texas red, tetrarhodimineisothiocyanate (TRITC), etc.), digoxigenin, biotin, phycoerythrin, AMCA,CyDyes™, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C or ³²P),enzymes (e.g. horseradish peroxidase, alkaline phosphotase, etc.),spectral calorimetric labels such as colloidal gold and colored glass orplastic (e.g. polysytrene, polypropylene, latex, etc.) beads. The choiceof label depends on sensitivity required, ease of conjugation with thecompound, stability requirements, and available instrumentation.

In general, a detector that monitors a particular probe or probecombination is used to detect the recognition reagent label. Typicaldetectors include spectrophotometers, phototubes and photodiodes,microscopes, scintillation counters, cameras, film and the like, as wellas combinations thereof. Examples of suitable detectors are widelyavailable from a variety of commercial sources known to persons ofskill.

High-Throughput Screening of Candidate Agents that Modulate PotassiumChannel Proteins

Conventionally, new chemical entities with useful properties aregenerated by identifying a chemical compound (called a “lead compound”)with some desirable property or activity, creating variants of the leadcompound, and evaluating the property and activity of those variantcompounds. However, the current trend is to shorten the time scale forall aspects of drug discovery. Because of the ability to test largenumbers quickly and efficiently, high throughput screening (HTS) methodsare replacing conventional lead compound identification methods.

In one preferred embodiment, high throughput screening methods involveproviding a library containing a large number of potential therapeuticcompounds (candidate compounds). Such “combinatorial chemical libraries”are then screened in one or more assays, as described herein, toidentify those library members (particular chemical species orsubclasses) that display a desired characteristic activity. Thecompounds thus identified can serve as conventional “lead compounds” orcan themselves be used as potential or actual therapeutics.

Combinatorial Chemical Libraries

Combinatorial chemical libraries are a preferred means to assist in thegeneration of new chemical compound leads. A combinatorial chemicallibrary is a collection of diverse chemical compounds generated byeither chemical synthesis or biological synthesis by combining, a numberof chemical “building blocks” such as reagents. For example, a linearcombinatorial chemical library such as a polypeptide library is formedby combining a set of chemical building blocks called amino acids inevery possible way for a given compound length (i.e., the number ofamino acids in a polypeptide compound). Millions of chemical compoundscan be synthesized through such combinatorial mixing of chemicalbuilding blocks. For example, one commentator has observed that thesystematic, combinatorial mixing of 100 interchangeable chemicalbuilding blocks results in the theoretical synthesis of 100 milliontetrameric compounds or 10 billion pentameric compounds (Gallop et al.(1994) 37(9): 12331250).

Preparation and screening of combinatorial chemical libraries are wellknown to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, e.g.,U.S. Pat. No. 5,010,175, Furka (1991) Int. J. Pept. Prot. Res., 37:487-493. Houghton et al. (1991) Nature, 354: 84-88). Peptide synthesisis by no means the only approach envisioned and intended for use withthe present invention. Other chemistries for generating chemicaldiversity libraries can also be used. Such chemistries include, but arenot limited to: peptoids (PCT Publication No. WO 91/19735, 26 Dec.1991), encoded peptides (PCT Publication WO 93/20242, 14 Oct. 1993),random biooligomers (PCT Publication WO 92/00091, 9 Jan. 1992),benzodiazepines (U.S. Pat. No. 5,288,514), diversomers such ashydantoins, benzodiazepines and dipeptides (Hobbs et al., (1993) Proc.Nat. Acad. Sci. USA 90: 69096913), vinylogous polypeptides (Hagihara etal. (1992) J. Amer. Chem. Soc. 114: 6568), nonpeptidal peptidomimeticswith a Beta D Glucose scaffolding (Hirschmann et al. (1992) J. Amer.Chem. Soc. 114: 92179218), analogous organic syntheses of small compoundlibraries (Chen et al. (1994) J. Amer. Chem. Soc. 116: 2661),oligocarbamates (Cho. et al., (1993) Science 261:1303), and/or peptidylphosphonates (Campbell et al., (1994) J. Org. Chem. 59: 658). See,generally, Gordon et al., (1994) J. Med. Chem. 37:1385, nucleic acidlibraries, peptide nucleic acid libraries (see, e.g. U.S. Pat. No.5,539,083) antibody libraries (see, e.g., Vaughn et al. (1996) NatureBiotechnology, 14(3): 309-314), and PCT/US96/10287), carbohydratelibraries (see, e.g. Liang et al. (1996) Science, 274: 1520-1522, andU.S. Pat. No. 5,593,853), and small organic molecule libraries (see,e.g., benzodiazepines, Baum (1993) C&EN, January 18, page 33,isoprenoids U.S. Pat. No. 5,569,588, thiazolidinones and metathiazanonesU.S. Pat. No. 5,549,974, pyrrolidines U.S. Pat. Nos. 5,525,735 and5,519,134, morpholino compounds U.S. Pat. No. 5,506,337, benzodiazepinesU.S. Pat. No. 5,288,514, and the like).

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 MPS, 390 MPS. Advanced Chem Tech, LouisvilleKy., Symphony. Rainin, Woburn, Mass., 433A Applied Biosystems, FosterCity, Calif., 9050 Plus, Millipore. Bedford, Mass.).

A number of well known robotic systems have also been developed forsolution phase chemistries. These systems include automated workstationslike the automated synthesis apparatus developed by Takeda ChemicalIndustries, LTD. (Osaka, Japan) and many robotic systems utilizingrobotic arms (Zymate II, Zymark Corporation, Hopkinton, Mass.; Orca,HewlettPackard, Palo Alto, Calif.) which mimic the manual syntheticoperations performed by a chemist. Any of the above devices are suitablefor use with the present invention. The nature and implementation ofmodifications to these devices (if any) so that they can operate asdiscussed herein will be apparent to persons skilled in the relevantart. In addition, numerous combinatorial libraries are themselvescommercially available (see, e.g., ComGenex, Princeton. N.J., Asinex,Moscow, Ru, Tripos, Inc., St. Louis, Mo., ChemStar, Ltd. Moscow, RU, 3DPharmaceuticals, Exton, Pa. Martek Biosciences, Columbia, Md., etc.).

High Throughput Assays of Chemical Libraries

Any of the assays for compounds capable of modulating potassium ionchannel proteins described herein are amenable to high throughputscreening. High throughput screening systems are commercially available(see, e.g., Zymark Corp., Hopkinton, Mass.; Air Technical Industries,Mentor, Ohio; Beckman Instruments, Inc. Fullerton. CA; PrecisionSystems, Inc., Natick, Mass., etc.). These systems typically automateentire procedures including all sample and reagent pipetting, liquiddispensing, timed incubations, and final readings of the microplate indetector(s) appropriate for the assay. These configurable systemsprovide high thruput and rapid start up as well as a high degree offlexibility and customization. The manufacturers of such systems providedetailed protocols the various high throughput. Thus, for example,Zymark Corp. provides technical bulletins describing screening systemsfor detecting the modulation of gene transcription, ligand binding, andthe like.

Assays for Modulation of Potassium Flow

The activity of functional potassium channels of this invention can beassessed using a variety of in vitro and in vivo assays, e.g., measuringvoltage, current, measuring membrane potential, measuring ion flux,e.g., potassium or rubidium, measuring potassium concentration,measuring second messengers and transcription levels, and using e.g.,voltage-sensitive dyes, radioactive tracers, and patch-clampelectrophysiology. In particular such assays can be used to test formodulators both inhibitors and activators of channels.

Modulators of the potassium channels are tested using biologicallyactive, functional two-transmembrane domain type potassium ion channels,either recombinant or naturally occurring. In recombinantly basedassays, the subunits are typically expressed and modulation is testedusing one of the in vitro or in vivo assays described below.

In brief, samples or assays that are treated with a potential channelinhibitors or activators are compared to control samples without thetest compound, to examine the extent of modulation. Control samplese.g., those untreated with activators or inhibitors are assigned arelative potassium channel activity value of 100. Inhibition is presentwhen potassium channel activity value relative to the control is about90%, preferably 50%, more preferably 25%. Activation of channels isachieved when the select potassium channel activity value relative tothe control is 110%, more preferably 150%, more preferable 200% higher.

Changes in ion flux may be assessed by determining changes inpolarization (i.e., electrical potential) of the cell or membraneexpressing the potassium channels of this invention A preferred means todetermine changes in cellular polarization is by measuring changes incurrent (thereby measuring changes in polarization) with voltage-clampand patch-clamp techniques, e.g., the “cell-attached” mode, the“inside-out” mode, and the “whole cell” mode (see, e.g., Ackerman etal., New Engl. J. Med. 336:1575-1595 (1997)). Whole cell currents areconveniently determined using the standard methodology (see, e.g., Hamilet al., PFlugers. Archiv. 391:85 (1981). Other known assays include:radiolabeled rubidium flux assays and fluorescence assays usingvoltage-sensitive dyes (see, e.g. Vestergarrd-Bogind et al., J MembraneBiol. 88:67-75 (1988); Daniel et al., J. Pharmacol. Meth. 25: 185-193(1991): Holevinsky et al. J. Membrane Biology 137:59-70 (1994)). Assaysfor compounds capable of inhibiting or increasing potassium flux throughthe channel proteins can be performed by application of the compounds toa bath solution in contact with and comprising cells having an channelof the present invention (see, e.g., Blatz et al., Nature 323:718-720(1986); Park, J. Physiol. 481:555-570 (1994)). Generally, the compoundsto be tested are present in the range from 1 pM to 100 mM.

The effects of the test compounds upon the function of the channels canbe measured by changes in the electrical currents or ionic flux or bythe consequences of changes in currents and flux. Changes in electricalcurrent or ionic flux are measured by either increases or decreases influx of cations such as potassium or rubidium ions. The cations can bemeasured in a variety of standard ways. They can be measured directly byconcentration changes of the ions or indirectly by membrane potential orby radiolabeling of the ions. Consequences of the test compound on ionflux can be quite varied. Accordingly, any suitable physiological changecan be used to assess the influence of a test compound on the channelsof this invention. The effects of a test compound can be measured by atoxin binding assay. When the functional consequences are determinedusing intact cells or animals, one can also measure a variety of effectssuch as transmitter release (e.g., dopamine), hormone release (e.g.,insulin), transcriptional changes to both known and uncharacterizedgenetic markers (e.g., northern blots), cell volume changes (e.g., inred blood cells), immunoresponses (e.g., T cell activation), changes incell metabolism such as cell growth or pH changes, and changes inintracellular second messengers such as [Ca²⁺].

Prokaryotic Cation Channel Protein Mutated to Mimic a FunctionalEukaryotic Cation Channel Protein

Furthermore, as explained above, the present invention extends toprokaryotic cation channel proteins mutated to mimic a functionaleukaryotic cation channel protein. These mutated cation channel proteinshave broad applications in assays for screening potential drugs ortherapeutic agents which potentially can interact with eukaryotic cationchannel proteins, and be used to treat numerous conditions related tothe function of cation channel proteins in vivo, such as cardiacarrhythmia, diabetes mellitus, seizure disorder, asthma or hypertension,to name only a few.

Presently available recombinant DNA techniques, such as site directedmutagenesis for example, can be used to readily mutate one or a numberof codons of an isolated nucleic acid molecule encoding. A prokaryoticcation channel protein which can then be expressed to produce a mutatedprokaryotic cation channel protein which mimics a eukaryotic cationchannel protein.

Furthermore, prokaryotic cation channel proteins having applications inthis aspect of the present invention comprise prokaryotic potassiumchannel proteins, prokaryotic sodium channel proteins, or prokaryoticcalcium channel proteins. Such prokaryotic cation channel proteins canbe obtained from varying prokaryotic organisms, such as E. coli,Streptomyces lividans, Clostridium acetobutylicum, or Staphylcoccusaureus, to name only a few. More specifically, a prokaryotic potassiumchannel protein comprising an amino acid sequence of SEQ ID NOs:1, 2, 3,or 7, or conserved variants thereof, can be mutated to mimic thephysiological functions and chemical properties of numerous eukaryoticcation channel proteins. In a preferred embodiment, a potassium channelprotein from Streptomyces lividans is mutated to mimic the physiologicalfunctions and chemical properties of a eukaryotic cation channelprotein, such as a eukaryotic potassium channel protein, a eukaryoticsodium channel protein, or a eukaryotic calcium channel protein.Consequently, a potential drug or agent which interacts with a mutatedprokaryotic channel protein of the present invention, such as bindingthereto for example, should undergo the same or similar interactionswith a eukaryotic cation channel protein the prokaryotic cation channelprotein was mutated to mimic. Hence, a mutated prokaryotic cationchannel protein of the present invention can serve as a model for aspecific eukaryotic cation channel protein in screening potential drugsor therapeutic agents for interaction therewith.

Moreover, pursuant to the present invention, and using recombinant DNAtechniques, a prokaryotic cation channel protein can be mutated to mimiceukaryotic cation channel proteins from numerous eukaryotic organisms,such as, for example, insects or mammals. More specifically, aprokaryotic cation channel protein can be mutated to mimic eukaryoticcation channel proteins from a wide variety of eukaryotic organisms,such as Drosophila melanogaster, Homo sapiens, C. elegans, Mus musculus,Arabidopsis thaliana, or Rattus novegicus, to name only a few. Sucheukaryotic cation channel proteins comprise an amino acid sequencecomprising SEQ ID Nos: 4, 5, 6, 8, 9, 10, 11, 12, 13, or 14, orconserved variants thereof.

In a preferred embodiment of the present invention, the prokaryoticcation channel protein comprises a potassium channel protein fromStreptomyces lividans comprising an amino acid sequence of SEQ ID NO:1,or conserved variants thereof, which is mutated to comprise an aminoacid sequence of SEQ ID NO:16, or conserved variants thereof, in orderto mimic the physiological functions and chemical properties of aeukaryotic cation channel protein comprising an amino acid sequence ofSEQ ID NO:4. Moreover, such a mutated prokaryotic cation channel proteinof the present invention is encoded by an isolated nucleic acid moleculecomprising a DNA sequence of SEQ ID NO:17, or degenerate variantsthereof.

Mutant Cation Channel Protein

Moreover, the present invention is directed to a mutant cation channelprotein. More specifically, the present invention comprises a mutantpotassium channel protein comprising an amino acid sequence of SEQ IDNO:16, or conserved variants thereof.

The nomenclature used to define the polypeptides is that specified bySchroder & Lubke, “The Peptides”, Academic Press (1965), wherein inaccordance with conventional representation the amino group at theN-terminal appears to the left and the carboxyl group at the C-terminalto the right. NH₂, refers to the amide group present at the carboxyterminus when written at the right of a polypeptide sequence.

Accordingly, conserved variants of an isolated mutant cation channelprotein of the present invention displaying substantially equivalentactivity to an isolated cation channel protein of the present invention,are likewise contemplated for use in the present invention. Thesemodifications can be obtained through peptide synthesis utilizing theappropriate starting material.

In keeping with standard polypeptide nomenclature, J. Biol. Chem.,243:3552-59 (1969), abbreviations for amino acid residues are shown inthe following Table of Correspondence: TABLE OF CORRESPONDENCE SYMBOL1-Letter 3-Letter AMINO ACID Y Tyr tyrosine G Gly glycine F Phephenylalanine M Met methionine A Ala alanine S Ser serine I Ileisoleucine L Leu leucine T Thr threonine V Val valine P Pro proline KLys lysine H His histidine Q Gln glutamine E Glu glutamic acid W Trptryptophan R Arg arginine D Asp aspartic acid N Asn asparagine C Cyscysteine

It should be noted that all amino-acid residue sequences are representedherein by formulae whose left and right orientation is in theconventional direction of amino-terminus to carboxy-terminus.Furthermore, it should be noted that a dash at the beginning or end ofan amino acid residue sequence indicates a peptide bond to a furthersequence of one or more amino-acid residues. The above Table ispresented to correlate the three-letter and one-letter notations whichmay appear alternately herein.

Hence, an amino acid in the mutant cation channel protein of the presentinvention can be changed in a non-conservative manner (i.e., by changingan amino acid belonging to a grouping of amino acids having a particularsize or characteristic to an amino acid belonging to another grouping)or in a conservative manner (i.e., by changing an amino acid belongingto a grouping of amino acids having a particular size or characteristicto an amino acid belonging to the same grouping). Such a conservativechange generally leads to less change in the structure and function ofthe resulting polypeptide. The present invention should be considered toinclude analogs whose sequences contain conservative changes which donot significantly alter the activity or binding characteristics of theresulting polypeptide.

The following is one example of various groupings of amino acids:

Amino Acids with Nonpolar R Groups

-   Alanine-   Valine-   Leucine-   Isoleucine-   Proline-   Phenylalanine-   Tryptophan-   Methionine    Amino Acids with Uncharged Polar R Groups-   Glycine-   Serine-   Threonine-   Cysteine-   Tyrosine-   Asparagine-   Glutamine    Amino Acids with Charged Polar R Groups (Negatively Charged at pH    6.0)-   Aspartic acid-   Glutamic acid    Basic Amino Acids (Positively Charged at pH 6.0)-   Lysine-   Arginine-   Histidine (at pH 6.0)    Another grouping may be those amino acids with aromatic groups:-   Phenylalanine-   Tryptophan-   Tyrosine    Another grouping may be according to molecular weight (i.e., size of    R groups):-   Glycine 75-   Alanine 89-   Serine 105-   Proline 115-   Valine 117-   Threonine 119-   Cysteine 121-   Leucine 131-   Isoleucine 131-   Asparagine 132-   Aspartic acid 133-   Glutamine 146-   Lysine 146-   Glutamic acid 147-   Methionine 149-   Histidine (at pH 6.0) 155-   Phenylalanine 165-   Arginine 174-   Tyrosine 181-   Tryptophan 204    Particularly preferred substitutions are:    -   Gin for Arg or Lys; and    -   His for Lys or Arg.

Amino acid substitutions may also be introduced to substitute an aminoacid with a particularly preferable property. For example, a Cys may beintroduced a potential site for disulfide bridges with another Cys, orwith a carrier of the present invention. A His may be introduced as aparticularly “catalytic” site (i.e., His can act as an acid or base andis the most common amino acid in biochemical catalysis). Pro may beintroduced because of its particularly planar structure, which inducesβ-turns in the polypeptide's structure. Alternately, D-amino acids canbe substituted for the L-amino acids at one or more positions.

Antibodies to an Isolated Mutant Cation Channel Protein of the Invention

As explained above, the present invention further extends to antibodiesof a cation channel protein of the present invention, or conservedvariants thereof. Such antibodies include but are not limited topolyclonal, monoclonal, chimeric, single chain, Fab fragments, and anFab expression library. The anti-mutant channel cation proteinantibodies of the invention may be cross reactive, e.g., they mayrecognize cation channel proteins from different species, and evendifferent types of cation channel proteins, i.e. potassium, sodium,calcium channel proteins, or their numerous variants which are gatedwith different mechanisms (i.e. voltage-gated, mechanical gated, ligandbinding gated, etc.). Polyclonal antibodies have greater likelihood ofcross reactivity.

Various procedures known in the art may be used for the production ofpolyclonal antibodies to an isolated mutant cation channel protein, orconserved variants thereof, of the present invention. For the productionof antibody, various host animals can be immunized by injection with amutant cation channel protein, or conserved variants thereof, includingbut not limited to rabbits, mice, rats, sheep, goats, etc. Furthermore,a mutant cation channel protein, or conserved variants thereof, of thepresent invention, may be conjugated to an immunogenic carrier, e.g.,bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH). Variousadjuvants may be used to increase the immunological response, dependingon the host species, including but not limited to Freund's (complete andincomplete), mineral gels such as aluminum hydroxide, surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanins, dinitrophenol, andpotentially useful human adjuvants such as BCG (bacille Calmette-Guerin)and Corynebacterium parvum.

For preparation of monoclonal antibodies directed toward a mutant cationchannel protein of the present invention, or conserved variants thereof,any technique that provides for the production of antibody molecules bycontinuous cell lines in culture may be used. These include but are notlimited to the hybridoma technique originally developed by Kohler andMilstein [Nature 256:495-497 (1975)], as well as the trioma technique,the human B-cell hybridoma technique [Kozbor et al., Immunology Today4:72 1983): Cote et al., Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030(1983)], and the EBV-hybridoma technique to produce human monoclonalantibodies [Cole et al., in Monoclonal Antibodies and Cancer Therapy,Alan R. Liss. Inc., pp. 77-96 (1985)]. In an additional embodiment ofthe invention, monoclonal antibodies can be produced in germ-freeanimals utilizing recent technology [PCT/US90/02545]. In fact, accordingto the invention, techniques developed for the production of “chimericantibodies” [Morrison et al., J. Bacteriol. 159:870 (1984); Neuberger etal., Nature 312:604-608 (1984); Takeda et al., Nature 314:452454 (1985)]by splicing the genes from a mouse antibody molecule specific for anisolated mutant cation channel protein of the present invention, orconserved variants thereof, together with a fragment of a human antibodymolecule of appropriate biological activity can be used; such antibodiesare within the scope of this invention.

According to the invention, techniques described for the production ofsingle chain antibodies [U.S. Pat. Nos. 5,476,786 and 5,132,405 toHuston; U.S. Pat. No. 4,946,778] can be adapted to produce single chainantibodies specific for an isolated mutant cation channel protein of theinvention or conserved variants thereof. An additional embodiment of theinvention utilizes the techniques described for the construction of Fabexpression libraries [Huse et al. Science 246:1275-1281 (1989)] to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity for an isolated mutant cation channel protein of thepresent invention, or conserved variants thereof.

Antibody fragments which contain the idiotype of the antibody moleculecan be generated by known techniques. For example, such fragmentsinclude but are not limited to: the F(ab′)₂ fragment which can beproduced by pepsin digestion of the antibody molecule; the Fab′fragments which can be generated by reducing the disulfide bridges ofthe F(ab′)₂, fragment, and the Fab fragments which can be generated bytreating the antibody molecule with papain and a reducing agent.

In the production of antibodies, screening for the desired antibody canbe accomplished by techniques known in the art, e.g., radioimmunoassay,ELISA (enzyme-linked immunosorbant assay), “sandwich” immunoassays,immunoradiometric assays, gel diffusion precipitin reactions,immunodiffusion assays, in situ immunoassays (using colloidal gold,enzyme or radioisotope labels, for example), western blots,precipitation reactions, agglutination assays (e.g., gel agglutinationassays, hemagglutination assays), complement fixation assays,immunofluorescence assays, protein A assays, and immunoelectrophoresisassays, etc. In one embodiment, antibody binding is detected bydetecting a label on the primary antibody. In another embodiment, theprimary antibody is detected by detecting binding of a secondaryantibody or reagent to the primary antibody. In a further embodiment,the secondary antibody is labeled. Many means are known in the art fordetecting binding in an immunoassay and are within the scope of thepresent invention. For example, to select antibodies which recognize aspecific epitope of an isolated mutant cation channel protein of thepresent invention, or conserved variants thereof, one may assaygenerated hybridomas for a product which binds to a fragment of anisolated mutant cation channel protein, or conserved variants thereof,containing such epitope. For selection of an

The foregoing antibodies can be used in methods known in the artrelating to the localization and activity of an isolated mutant cationchannel protein, or conserved variants thereof, e.g., for Westernblotting, imaging such a cation channel protein in situ, measuringlevels thereof in appropriate physiological samples, etc. using any ofthe detection techniques mentioned above or known in the art.

In a specific embodiment, antibodies that agonize or antagonize theactivity of an isolated mutant cation channel protein of the presentinvention, or conserved variants thereof, can be generated. Suchantibodies can be tested using the assays described infra foridentifying ligands.

Detectably Labeled Antibodies of an Isolated Mutant Cation ChannelProtein of the Present Invention, or Conserved Variants Thereof

Moreover, the present invention extends to antibodies described above,detectably labeled. Suitable detectable labels include enzymes,radioactive isotopes, fluorophores (e.g., fluorescene isothiocyanate(FITC), phycoerythrin (PE), Texas red (TR), rhodamine, free or chelatedlanthanide series salts, especially Eu³⁺, to name a few fluorophores),chromophores, radioisotopes, chelating agents, dyes, colloidal gold,latex particles, ligands (e.g., biotin), and chemiluminescent agents.When a control marker is employed, the same or different labels may beused for the receptor and control marker.

In the instance where a radioactive label, such as the isotopes ³H, ¹⁴C,³²P, ³⁵S, ³⁶Cl, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ¹²⁵I, ¹³¹I, and ¹⁸⁶Re areused, known currently available counting procedures may be utilized. Inthe instance where the label is an enzyme, detection may be accomplishedby any of the presently utilized colorimetric, spectrophotometric,fluorospectrophotometric, amperometric or gasometric techniques known inthe art.

Direct labels are one example of labels which can be used according tothe present invention. A direct label has been defined as an entity,which in its natural state, is readily visible, either to the naked eye,or with the aid of an optical filter and/or applied stimulation, e.g.U.V. light to promote fluorescence. Among examples of colored labels,which can be used according to the present invention, include metallicsol particles, for example, gold sol particles such as those describedby Leuvering (U.S. Pat. No. 4,313,734): dye sole particles such asdescribed by Gribnau et al. (U.S. Pat. No. 4,373,932) and May et al. (WO88/08534): dyed latex such as described by May, supra, Snyder (EP-A 0280 559 and 0 281 327); or dyes encapsulated in liposomes as describedby Campbell et al. (U.S. Pat. No. 4,703,017). Other direct labelsinclude a radionucleotide, a fluorescent moiety or a luminescent moiety.In addition to these direct labeling devices, indirect labels comprisingenzymes can also be used according to the present invention. Varioustypes of enzyme linked immunoassays are well known in the art, forexample, alkaline phosphatase and horseradish peroxidase, lysozyme,glucose-6-phosphate dehydrogenase, lactate dehydrogenase, urease, theseand others have been discussed in detail by Eva Engvall in EnzymeImmunoassay ELISA and EMIT in Methods in Enzymology, 70, 419-439, 1980and in U.S. Pat. No. 4,857,453.

Suitable enzymes include, but are not limited to, alkaline phosphataseand horseradish peroxidase.

Other labels for use in the invention include magnetic beads or magneticresonance imaging labels.

As explained above, the present invention contemplates an isolatednucleic molecule, or degenerate variants thereof, which encode a mutantcation channel protein, or conserved variants thereof. Accordingly, withthe present invention, there may be employed conventional molecularbiology, microbiology, and recombinant DNA techniques within the skillof the art. Such techniques are explained fully in the literature. See,e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A LaboratoryManual, Second Edition (1989) Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (herein “Sambrook et al., 1989”): DNA Cloning: APractical Approach, Volumes I and II (D. N. Glover ed. 1985);Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic AcidHybridization[B. D. Hames & S. J. Higgins eds. (1985)]; TranscriptionAnd Translation[B. D. Hames & S. J. Higgins, eds. (1984)]; Animal CellCulture[R. I. Freshney, ed. (1986)]; Immobilized Cells And Enzymes[IRLPress, (1986)]; B. Perbal, A Practical Guide To Molecular Cloning(1984): F. M. Ausubel et al. (eds.), Current Protocols in MolecularBiology, John Wiley & Sons. Inc. (1994).

Therefore, if appearing herein, the following terms shall have thedefinitions set out below.

A “vector” is a replicon, such as plasmid, phage or cosmid, to whichanother DNA segment may be attached so as to bring about the replicationof the attached segment. A “replicon” is any genetic element (e.g.,plasmid, chromosome, virus) that functions as an autonomous unit of DNAreplication in vivo, i.e., capable of replication under its own control.

A “cassette” refers to a segment of DNA that can be inserted into avector at specific restriction sites. The segment of DNA encodes apolypeptide of interest, and the cassette and restriction sites aredesigned to ensure insertion of the cassette in the proper reading framefor transcription and translation.

A cell has been “transfected” by exogenous or heterologous DNA when suchDNA has been introduced inside the cell. A cell has been “transformed”by exogenous or heterologous DNA when the transfected DNA effects aphenotypic change. Preferably, the transforming DNA should be integrated(covalently linked) into chromosomal DNA making up the genome of thecell.

“Heterologous” DNA refers to DNA not naturally located in the cell, orin a chromosomal site of the cell. Preferably, the heterologous DNAincludes a gene foreign to the cell.

A “nucleic acid molecule” refers to the phosphate ester polymeric formof ribonucleosides (adenosine, guanosine, uridine or cytidine: “RNAmolecules”) or deoxyribonucleos ides (deoxyadenosine, deoxyguanosine,deoxythymidine, or deoxycytidine: “DNA molecules”), or any phosphoesteranalogs thereof, such as phosphorothioates and thioesters, in eithersingle stranded form, or a double-stranded helix. Double strandedDNA-DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic acidmolecule, and in particular DNA or RNA molecule, refers only to theprimary and secondary structure of the molecule, and does not limit itto any particular tertiary forms. Thus, this term includesdouble-stranded DNA found, inter alia, in linear or circular DNAmolecules (e.g., restriction fragments), plasmids, and chromosomes. Indiscussing the structure of particular double-stranded DNA molecules,sequences may be described herein according to the normal convention ofgiving only the sequence in the 5′ to 3′ direction along thenontranscribed strand of DNA (i.e., the strand having a sequencehomologous to the mRNA). A “recombinant DNA molecule” is a DNA moleculethat has undergone a molecular biological manipulation.

A nucleic acid molecule is “hybridizable” to another nucleic acidmolecule, such as a cDNA, genomic DNA, or RNA, when a single strandedform of the nucleic acid molecule can anneal to the other nucleic acidmolecule under the appropriate conditions of temperature and solutionionic strength (see Sambrook et al., supra). The conditions oftemperature and ionic strength determine the “stringency” of thehybridization. For preliminary screening for homologous nucleic acids,low stringency hybridization conditions, corresponding to a T_(m) of55°, can be used, e.g., 5×SCC, 0.1% SDS, 0.25% milk, and no formamide;or 30% formamide, 5×SCC, 0.5% SDS). Moderate stringency hybridizationconditions correspond to a higher T_(m), e.g., 40% formamide, with 5× or6×SCC. High stringency hybridization conditions correspond to thehighest T_(m), e.g., 50% formamide, 5× or 6× SCC. Hybridization requiresthat the two nucleic acids contain complementary sequences, althoughdepending on the stringency of the hybridization, mismatches betweenbases are possible. The appropriate stringency for hybridizing nucleicacids depends on the length of the nucleic acids and the degree ofcomplementation, variables well known in the art. The greater the degreeof similarity or homology between two nucleotide sequences, the greaterthe value of T_(m) for hybrids of nucleic acids having those sequences.The relative stability (corresponding to higher T_(m)) of nucleic acidhybridizations decreases in the following order: RNA:RNA, DNA:RNA,DNA:DNA. For hybrids of greater than 100 nucleotides in length,equations for calculating T_(m) have been derived (see Sambrook et al.,supra, 9.50-0.51). For hybridization with shorter nucleic acids, i.e.,oligonucleotides, the position of mismatches becomes more important, andthe length of the oligonucleotide determines its specificity (seeSambrook et al., supra, 11.7-11.8). Preferably a minimum length for ahybridizable nucleic acid is at least about 12 nucleotides; preferablyat least about 18 nucleotides; and more preferably the length is atleast about 27 nucleotides: and most preferably 36 nucleotides.

In a specific embodiment, the term “standard hybridization conditions”refers to a T_(m) of 55° C., and utilizes conditions as set forth above.In a preferred embodiment, the T_(m) is 60° C.; in a more preferredembodiment, the T_(m) is 65° C.

A DNA “coding sequence” is a double-stranded DNA sequence which istranscribed and translated into a polypeptide in a cell in vitro or invivo when placed under the control of appropriate regulatory sequences.The boundaries of the coding sequence are determined by a start codon atthe 5′ (amino) terminus and a translation stop codon at the 3′(carboxyl) terminus. A coding sequence can include, but is not limitedto, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNAsequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNAsequences. If the coding sequence is intended for expression in aeukaryotic cell, a polyadenylation signal and transcription terminationsequence will usually be located 3′ to the coding sequence.

Transcriptional and translational control sequences are DNA regulatorysequences, such as promoters, enhancers, terminators, and the like, thatprovide for the expression of a coding sequence in a host cell. Ineukaryotic cells, polyadenylation signals are control sequences.

A “promoter sequence” or “promoter” is a DNA regulatory region capableof binding RNA polymerase in a cell and initiating transcription of adownstream (3′ direction) coding sequence. For purposes of defining thepresent invention, the promoter sequence is bounded at its 3′ terminusby the transcription initiation site and extends upstream (5′ direction)to include the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlydefined for example, by mapping with nuclease S1), as well as proteinbinding domains (consensus sequences) responsible for the binding of RNApolymerase.

A coding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then trans-RNAspliced and translated into the protein encoded by the coding sequence.

As used herein, the term “sequence homology” in all its grammaticalforms refers to the relationship between proteins that possess a “commonevolutionary origin,” including proteins from superfamilies (e.g., theimmunoglobulin superfamily) and homologous proteins from differentspecies (e.g., myosin light chain, etc.) [Reeck et al., Cell, 50:667(1987)].

Accordingly, the term “sequence similarity” in all its grammatical formsrefers to the degree of identity or correspondence between nucleic acidor amino acid sequences of proteins that do not share a commonevolutionary origin [see Reeck et al., 1987, supra]. However, in commonusage and in the instant application, the term “homologous” whenmodified with an adverb such as “highly,” may refer to sequencesimilarity and not a common evolutionary origin.

In a specific embodiment, two DNA sequences are “substantiallyhomologous” or “substantially similar” when at least about 50%(preferably at least about 75%, and most preferably at least about 90 or95%) of the nucleotides match over the defined length of the DNAsequences. Sequences that are substantially homologous can be identifiedby comparing the sequences using standard software available in sequencedata banks, or in a Southern hybridization experiment under, forexample, stringent conditions as defined for that particular system.Defining appropriate hybridization conditions is within the skill of theart. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II,supra, Nucleic Acid Hybridization, supra.

Similarly, in a particular embodiment, two amino acid sequences are“substantially homologous” or “substantially similar” when greater than30% of the amino acids are identical, or greater than about 60% aresimilar (functionally identical). Preferably, the similar or homologoussequences are identified by alignment using, for example, the GCG(Genetics Computer Group, Program Manual for the GCG Package, Version 7,Madison, Wis.) pileup program.

The term “corresponding to” is used herein to refer similar orhomologous sequences, whether the exact position is identical ordifferent from the molecule to which the similarity or homology ismeasured. Thus, the term “corresponding to” refers to the sequencesimilarity, and not the numbering of the amino acid residues ornucleotide bases.

Moreover, due to degenerate nature of codons in the genetic code, amutant cation channel protein of the present invention can be encoded bynumerous isolated nucleic acid molecules. “Degenerate nature” refers tothe use of different three-letter codons to specify a particular aminoacid pursuant to the genetic code. It is well known in the art that thefollowing codons can be used interchangeably to code for each specificamino acid:

-   Phenylalanine (Phe or F) UUU or UUC-   Leucine (Leu or L) UUA or UUG or CUU or CUC or CUA or CUG-   Isoleucine (Ile or I) AUU or AUC or AUA-   Methionine (Met or M) AUG-   Valine (Val or V) GUU or GUC of GUA or GUG-   Serine (Ser or S) UCU or UCC or UCA or UCG or AGU or AGC-   Proline (Pro or P) CCU or CCC or CCA or CCG-   Threonine (Thr or T) ACU or ACC or ACA or ACG-   Alanine (Ala or A) GCU or GCG or GCA or GCG-   Tyrosine (Tyr or Y) UAU or UAC-   Histidine (His or H) CAU or CAC-   Glutamine (Gln or Q) CAA or CAG-   Asparagine (Asn or N) AAU or AAC-   Lysine (Lys or K) AAA or AAG-   Aspartic Acid (Asp or D) GAU or GAC-   Glutamic Acid (Glu or E) GAA or GAG-   Cysteine (Cys or C) UGU or UGC-   Arginine (Arg or R) CGU or CGC or CGA or CGG or AGA or AGG-   Glycine (Gly or G) GGU or GGC or GGA or GGG-   Tryptophan (Trp or W) UGG-   Termination codon UAA (ochre) or UAG (amber) or UGA (opal)

It should be understood that the codons specified above are for RNAsequences. The corresponding codons for DNA have a T substituted for U.

Furthermore, the present invention extends to an isolated nucleic acidmolecule, or degenerate variants thereof encoding a mutant cationchannel protein, detectably labeled, and a detectably labeled isolatednucleic acid molecule hybridizable under standard hybridizationconditions to an isolated nucleic acid molecule, or degenerate variantsthereof, encoding a cation channel protein of the present invention.Suitable detectable labels include enzymes, radioactive isotopes,fluorophores (e.g., fluorescene isothiocyanate (FITC), phycoerythrin(PE), Texas red (TR), rhodamine, free or chelated lanthanide seriessalts, especially Eu³⁺, to name a few fluorophores), chromophores,radioisotopes, chelating agents, dyes, colloidal gold, latex particles,ligands (e.g., biotin), and chemiluminescent agents. When a controlmarker is employed, the same or different labels may be used for thereceptor and control marker.

In the instance where a radioactive label, such as the isotopes ³H, ¹⁴C,³²P, ³⁵S, ³⁶CL, ⁵¹CR, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ¹²⁵I, ¹³¹I, and ¹⁵⁶Re, areused, known currently available counting procedures may be utilized. Inthe instance where the label is an enzyme, detection may be accomplishedby any of the presently utilized calorimetric, spectrophotometric,fluorospectrophotometric, amperometric or gasometric techniques known inthe art.

Direct labels are one example of labels which can be used according tothe present invention. A direct label has been defined as an entity,which in its natural state, is readily visible, either to the naked eye,or with the aid of an optical filter and/or applied stimulation, e.g.U.V. light to promote fluorescence. Among examples of colored labels,which can be used according to the present invention, include metallicsot particles, for example, gold sot particles such as those describedby Leuvering (U.S. Pat. No. 4,313,734); dye sole particles such asdescribed by Gribnau et al. (U.S. Pat. No. 4,373,932) and May et al. (WO88/08534): dyed latex such as described by May, supra, Snyder (EP-A 0280 559 and 0 281 327); or dyes encapsulated in liposomes as describedby Campbell et al. (U.S. Pat. No. 4,703,017). Other direct labelsinclude a radionucleotide, a fluorescent moiety or a luminescent moiety.In addition to these direct labeling devices, indirect labels comprisingenzymes can also be used according to the present invention. Varioustypes of enzyme linked immunoassays are well known in the art, forexample, alkaline phosphatase and horseradish peroxidase, lysozyme,glucose-6-phosphate dehydrogenase, lactate dehydrogenase, urease, theseand others have been discussed in detail by Eva Engvall in EnzymeImmunoassay ELISA and EMIT in Methods in Enzymology, 70.419-439, 1980and in U.S. Pat. No. 4,857,453.

Suitable enzymes include, but are not limited to, alkaline phosphataseand horseradish peroxidase.

Other labels for use in the invention include magnetic beads or magneticresonance imaging labels.

Cloning Vectors

The present invention also extends to cloning vectors comprising anisolated nucleic acid molecule of the present invention, or degeneratevariants thereof, and an origin of replication. For purposes of thisApplication, an “origin of replication refers to those DNA sequencesthat participate in DNA synthesis.

As explained above, in an embodiment of the present invention, anisolated nucleic acid molecule, or degenerate variants thereof, encodinga mutant cation channel protein of the present invention, along withisolated nucleic acid molecules hybridizable under standardhybridization conditions to an isolated nucleic acid, or degeneratevariants thereof, which encodes a mutant cation channel protein of thepresent invention, can be inserted into an appropriate cloning vector inorder to produce multiple copies of the isolated nucleic acid. A largenumber of vector-host systems known in the art may be used. Possiblevectors include, but are not limited to, plasmids or modified viruses,but the vector system must be compatible with the host cell used.Examples of vectors include, but are not limited to, E. coli,bacteriophages such as lambda derivatives, or plasmids such as pBR322derivatives or pUC plasmid derivatives, e.g., pGEX vectors, pmal-c,pFLAG, etc. The insertion into a cloning vector can, for example, beaccomplished by ligating an isolated nucleic acid molecule of thepresent invention or degenerate variants thereof, or an isolated nucleicacid hybridizable thereto under standard hybridization conditions, intoa cloning vector which has complementary cohesive termini. However, ifthe complementary restriction sites used to fragment the isolatednucleic acid or degenerate variants thereof, or an isolated nucleic acidhybridizable thereto under standard hybridization conditions, are notpresent in the cloning vector, the ends of the isolated nucleic acidmolecule or degenerate variants thereof, or an isolated nucleic acidmolecule hybridizable under standard hybridization conditions theretomay be enzymatically modified. Alternatively, any site desired may beproduced by ligating nucleotide sequences (linkers) onto the DNAtermini; these ligated linkers may comprise specific chemicallysynthesized oligonucleotides encoding restriction endonucleaserecognition sequences. Such recombinant molecules can then be introducedinto host cells via transformation, transfection, infection,electroporation, etc., so that many copies of an isolated nucleic acidmolecule of the present invention, or degenerate variants thereof, or anan isolated nucleic acid molecule hybridizable thereto under standardhybridization conditions, can be generated. Preferably, the clonedisolated nucleic acid molecule is contained on a shuttle vector plasmid,which provides for expansion in a cloning cell, e.g., E. coli, andfacile purification for subsequent insertion into an appropriateexpression cell line, if such is desired. For example, a shuttle vector,which is a vector that can replicate in more than one type of organism,can be prepared for replication in both E. coli and Saccharomycescerevisiae by linking sequences from an E. coli plasmid with sequencesfrom the yeast 2μ plasmid.

In an alternative method, an isolated nucleic acid molecule of thepresent invention, or degenerate variants thereof, or an isolatednucleic acid molecule hybridizable thereto under standard hybridizationconditions may be identified and isolated after insertion into asuitable cloning vector in a “shot gun” approach. Enrichment for anisolated nucleic acid molecule, for example; by size fractionation, canbe done before insertion into the cloning vector.

Expression Vectors

As stated above, the present invention extends to an isolated nucleicacid molecule encoding a mutant cation channel protein of the presentinvention, degenerate variants thereof, or an isolated nucleic acidhybridizable thereto under standard hybridization conditions.

Isolated nucleic acid molecules of the present invention can be insertedinto an appropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedprotein-coding sequence. Such elements are termed herein a “promoter.”Thus, an isolated nucleic acid molecule, or degenerate variants thereof,which encodes a mutant cation channel protein of the present, along withisolated nucleic acid molecules hybridizable thereto under standardhybridization conditions is operatively associated with a promoter in anexpression vector of the invention. A DNA sequence is “operativelyassociated” to an expression control sequence, such as a promoter, whenthe expression control sequence controls and regulates the transcriptionand translation of that DNA sequence. The term “operatively associated”includes having an appropriate start signal (e.g., ATG) in front of theDNA sequence to be expressed and maintaining the correct reading frameto permit expression of the DNA sequence under the control of theexpression control sequence and production of the desired productencoded by the DNA sequence. If an isolated nucleic acid molecule of thepresent invention does not contain an appropriate start signal, such astart signal can be inserted into the expression vector in front of (5′of) the isolated nucleic acid molecule.

Both cDNA and genomic sequences can be cloned and expressed undercontrol of such regulatory sequences. An expression vector alsopreferably includes a replication origin.

The necessary transcriptional and translational signals can be providedon a recombinant expression vector, or they may be supplied by thenative gene encoding the wild type variant of a mutant cation channelprotein of the present invention, and/or its flanking regions.

Potential host-vector systems include but are not limited to mammaliancell systems infected with virus (e.g., vaccinia virus, adenovirus,etc.); insect cell systems infected with virus (e.g., baculovirus);microorganisms such as yeast containing yeast vectors; or bacteriatransformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. Theexpression elements of vectors vary in their strengths andspecificities. Depending on the host-vector system utilized, any one ofa number of suitable transcription and translation elements may be used.

Moreover, an isolated nucleic acid molecule of the present invention maybe expressed chromosomally, after integration of the coding sequence byrecombination. In this regard, any of a number of amplification systemsmay be used to achieve high levels of stable gene expression (SeeSambrook et al. 1989, supra).

A unicellular host containing a recombinant vector comprising anisolated nucleic acid molecule, or degenerate variants thereof, whichencodes a mutant cation channel protein of the present invention, or anisolated nucleic acid molecule hybridizable under standard hybridizationconditions to an isolated nucleic acid molecule, or degenerate variantsthereof, which encodes a mutant cation channel protein of the presentinvention, is cultured in an appropriate cell culture medium underconditions that provide for expression of the isolated nucleic acidmolecule by the cell.

Any of the methods previously described for the insertion of DNAfragments into a cloning vector may be used to construct expressionvectors comprising an isolated nucleic acid molecule of the presentinvention, and appropriate transcriptional/translational control signalsand the protein coding sequences. These methods may include in vitrorecombinant DNA and synthetic techniques and in vivo recombination(genetic recombination).

Expression of an isolated nucleic acid molecule of the presentinvention, degenerate variants thereof, or an isolated nucleic acidmolecule hybridizable thereto under standard hybridization conditions,along with a an isolated mutant cation channel protein encoded byisolated nucleic acid molecules of the present invention, degeneratevariants thereof, or an isolated nucleic acid molecule hybridizablethereto under standard hybridization conditions, may be controlled byany promoter/enhancer element known in the art, but these regulatoryelements must be functional in the host selected for expression.Promoters which may be used to control expression include, but are notlimited to, the SV40 early promoter region (Benoist and Chambon, 1981,Nature 290:304-310), the promoter contained in the 3′ long terminalrepeat of Rous sarcoma virus (Yamamoto, et al., 1980, Cell 22:787-797),the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl.Acad. Sci. U.S.A. 78:1441-1445), the regulatory sequences of themetallothionein gene (Brinster et al., 1982. Nature 296:3942):prokaryotic expression vectors such as the β-lactamase promoter(Villa-Kamaroff, et al., 1978, Proc. Natl. Acad. Sci. U.S.A.75:3727-3731), or the tac promoter (DeBoer, et al., 1983. Proc. Natl.Acad. Sci. U.S.A. 80:21-25); see also “Useful proteins from recombinantbacteria” in Scientific American, 1980, 242:74-94; promoter elementsfrom yeast or other fungi such as the Gal 4 promoter, the ADC (alcoholdehydrogenase) promoter. PGK (phosphoglycerol kinase) promoter, alkalinephosphatase promoter; and the animal transcriptional control regions,which exhibit tissue specificity and have been utilized in transgenicanimals: elastase I gene control region which is active in pancreaticacinar cells (Swift et al., 1984. Cell 38:639-646; Ornitz et al. 1986.Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987,Hepatology 7:425-515); insulin gene control region which is active inpancreatic beta cells (Hanahan, 1985, Nature 315:115-122),immunoglobulin gene control region which is active in lymphoid cells(Grosschedl et al., 1984, Cell 38:647-658; Adames et al., 1985, Nature318:533-538; Alexander et al., 1987, Mol. Cell. Biol. 7:1436-1444),mouse mammary tumor virus control region which is active in testicular,breast, lymphoid and mast cells (Leder et al., 1986. Cell 45:485-495),albumin gene control region which is active in liver (Pinkert et al.,1987, Genes and Devel. 1:268-276), alpha-fetoprotein gene control regionwhich is active in liver (Krumlauf et al. 1985, Mol. Cell. Biol.5:1639-1648; Hammer et al., 1987, Science 235:53-58), alpha1-antitrypsin gene control region which is active in the liver (Kelseyet al., 1987, Genes and Devel. 1:161-171), beta-globin gene controlregion which is active in myeloid cells (Mogram et al., 1985, Nature315:338-340; Kollias et al., 1986, Cell 46:89-94), myelin basic proteingene control region which is active in oligodendrocyte cells in thebrain (Readhead et al., 1987. Cell 48:703-712), myosin light chain-2gene control region which is active in skeletal muscle (Sani, 1985,Nature 314:283-286), and gonadotropic releasing hormone gene controlregion which is active in the hypothalamus (Mason et al. 1986, Science234:1372-1378).

Expression vectors comprising an isolated nucleic acid molecule, ordegenerate variants thereof, encoding a mutant cation channel protein ofthe present invention, or an expression vector comprising an isolatednucleic acid molecule hybridizable under standard hybridizationconditions to an isolated nucleic acid molecule of the presentinvention, can be identified by four general approaches: (a) PCRamplification of the desired plasmid DNA or specific mRNA, (b) nucleicacid hybridization, (c) presence or absence of selection marker genefunctions, and (d) expression of inserted sequences. In the firstapproach, the nucleic acids can be amplified by PCR to provide fordetection of the amplified product. In the second approach, the presenceof a foreign gene inserted in an expression vector can be detected bynucleic acid hybridization using probes comprising sequences that arehomologous to an inserted marker gene. In the third approach, therecombinant vector/host system can be identified and selected based uponthe presence or absence of certain “selection marker” gene functions(e.g., β-galactosidase activity, thymidine kinase activity, resistanceto antibiotics, transformation phenotype, occlusion body formation inbaculovirus, etc.) caused by the insertion of foreign genes in thevector. In another example, if an isolated nucleic of the presentinvention, or degenerate variants thereof, which encode a mutant cationchannel protein of the present invention or conserved variants thereof,or an isolated nucleic acid molecule hybridizable thereto under standardhybridization conditions, is inserted within the “selection marker” genesequence of the vector, recombinants containing the insert can beidentified by the absence of the inserted gene function. In the fourthapproach, recombinant expression vectors can be identified by assayingfor the activity, biochemical, or immunological characteristics of thegene product expressed by the recombinant, provided that the expressedprotein assumes a functionally active conformation.

Production of a Mutant Cation Channel Protein of the Present Invention

Moreover, the present invention extends to a method of producing amutant cation channel protein comprising an amino acid sequence of SEQID NO:16, or conserved variants thereof. More specifically, a method ofthe present invention comprises the steps of culturing a unicellularhost either transformed or transfected with an expression vector of thepresent invention explained above, under conditions that provide forexpression of the mutant cation channel protein, and recovering themutant cation channel protein from the transformed or transfectedunicellular host. As explained above, the conditions which provide forexpression of a mutant channel protein of the present invention aredependent upon the expression vector and promoter used to transform ortransfect a unicellular host of the invention. Since the conditionsneeded relative to the promoter used are within the knowledge of one ofordinary skill in this art, conditions for specific promoters are notrepeated here.

Moreover, collection of a cation channel protein of the presentinvention produced pursuant to the method stated above, is also withinthe knowledge of a skilled artisan.

Crystal of a Cation Channel Protein

As explained above, the present invention extends to a crystal of acation channel protein having a central pore, which is found natively ina lipid bilayer membrane of an animal cell, such that the central porecommunicates with extracellular matrix and cellular cytosol, wherein thecrystal effectively diffracts x-rays to a resolution of greater than 3.2angstroms.

Moreover, the present invention extends to a crystal of a cation channelprotein as described above, wherein the cation channel protein comprisesa first layer of aromatic amino acid residues positioned to extend intothe lipid bilayer membrane proximate to the interface an extracellularmatrix and lipid bilayer membrane, a second layer of aromatic amino acidresidues positioned to extend into the lipid bilayer membrane proximateto the interface of cellular cytosol and said lipid bilayer membrane, atetramer of four identical transmembrane subunits, and a central poreformed by the four identical transmembrane subunits.

Furthermore, each transmembrane subunit comprises an inner transmembranealpha-helix which has a kink therein, an outer transmembranealpha-helix, and a pore alpha-helix, wherein each subunit is insertedinto the tetramer of the cation channel protein so that the outertransmembrane helix of each subunit contacts the first and second layersof aromatic amino acid residues described above, and abuts the lipidbilayer membrane. Moreover, the inner transmembrane helix of eachsubunit abuts the central pore of the cation channel protein, contactsthe first and second layers of aromatic amino acid residues, is tiltedby about 25° with respect to the normal of the lipid bilayer membrane,and is packed against inner transmembrane alpha helices of othertransmembrane subunits at the second layer of aromatic amino acidresidues forming a bundle at the second layer. The pore alpha-helix ofeach subunit is located at the first layer of said aromatic amino acidresidues, and positioned between inner transmembrane alpha-helices ofadjacent subunits, and are directed, in an amino carboxyl sense, towardsa point near the center of the central pore.

It has been further determined, based on examination of a crystal of thepresent invention, that the central pore of a cation channel protein,comprises a pore region located at the first layer of aromatic aminoacid residues, and connected to the inner and outer transmembranealpha-helices of said subunits. More particularly, the pore regioncomprises about 25-45 amino acid residues, a turret connected to thepore alpha-helix and the outer alpha-helix, wherein the turret islocated at the interface of said extracellular matrix and the lipidbilayer membrane. The pore region further comprises an ion selectivityfilter connected to the pore alpha-helix and the inner transmembranealpha-helix of each subunit. The ion selectivity filter extends into thecentral pore of the cation channel protein, and comprises a signatureamino acid residue sequence having main chain atoms which create a stackof sequential oxygen atoms along the selectivity filter that extend intothe central pore, and amino acid residues having side chains thatinteract with the pore helix. It is the signature sequence which enablesa cation channel protein to discriminate among the cation intended topermeate the protein, and other cations, so that only the cationintended to permeate the channel protein is permitted to permeate.

The central pore further comprises a tunnel into the lipid bilayermembrane which communicates with the cellular cytosol, and a cavitylocated within the lipid bilayer membrane between the pore region andthe tunnel, and connected to the them, such that the central porecrosses the membrane.

Furthermore, the structure of all ion channel proteins share commonfeatures, which are set forth in the crystal of a cation channel proteindescribed above. Consequently, the present invention extends to acrystal of a cation channel protein having a central pore, which isdescribed above, wherein the cation is selected from the groupconsisting of: Na⁺, K⁺, and Ca²⁺. Hence, the present invention extendsto crystals of potassium channel proteins, sodium channel proteins, andcalcium ion channels, to name only a few. In a preferred embodiment, thecrystal of a cation channel protein comprises a crystal of a potassiumion channel protein.

In addition, a crystal of an ion channel protein of a present inventioncan comprise an amino acid sequence of any presently known, orsubsequently discovered cation protein channel. Consequently, thepresent invention extends to a crystal of a cation channel proteinhaving a central pore, which is found natively in a lipid bilayermembrane of an animal cell, such that the central pore communicates withextracellular matrix and cellular cytosol, wherein the crystal comprisesan amino acid sequence of:

-   -   residues 23 to 119 of SEQ ID NO:1 (Streptomyces lividans);    -   residues 61 to 119 of SEQ ID NO:2 (E. coli);    -   residues 61 to 119 of SEQ ID NO:3 (Clostridium acetobutylicum);    -   residues 61 to 119 of SEQ ID NO:4 (Drosophila melanogaster);    -   residues 61 to 119 of SEQ ID NO:5 (Homo sapiens);    -   residues 61 to 119 of SEQ ID NO:6 (Homo sapiens);    -   residues 61 to 119 of SEQ ID NO:7 (Paramecium tetraaurelia);    -   residues 61 to 119 of SEQ ID NO:8 (C. elegans);    -   residues 61 to 119 of SEQ ID NO:9 (Mus musculus);    -   residues 61 to 119 of SEQ ID NO:10 (Homo sapiens);    -   residues 61 to 119 of SEQ ID NO:11 (Arabidopsis thaliana);    -   residues 61 to 119 of SEQ ID NO:12 (Homo sapiens);    -   residues 61 to 119 of SEQ ID NO:13 (Rattus novegicus); or    -   residues 61 to 119 of SEQ ID NO:14 (Homo sapiens);        or conserved variants thereof.

In a preferred embodiment, a crystal of the present invention having acentral pore, which is found natively in a lipid bilayer membrane of ananimal cell, such that the central pore communicates with extracellularmatrix and cellular cytosol, comprises an amino sequence of amino acidresidues 23 to 119 of SEQ ID NO:1, has a space grouping of C2, and aunit cell of dimensions of a=128.8 Å, b=68.9 Å, c=112.0 Å, and β=124.6°.Moreover, preferably, the present invention extends to a crystal asdescribed above, wherein the cation K⁺.

Furthermore, the present invention extends to a crystal of a cationchannel protein having a central pore, which is found natively in alipid bilayer membrane of an animal cell, such that the central porecommunicates with extracellular matrix and cellular cytosol, wherein thechannel protein comprises a signature sequence comprising:

-   -   Thr-Val-Gly-Tyr-Gly-Asp (SEQ ID NO:15).

Method for Growing a Crystal of the Present Invention

The present invention further extends to a method for growing a crystalof a cation channel protein having a central pore, which is foundnatively in a lipid bilayer membrane of an animal cell, such that thecentral pore communicates with extracellular matrix and cellularcytosol, by sitting-drop vapor diffusion. Such a method of the presentinvention comprises the steps of providing the cation channel protein,removing a predetermined number of carboxy terminal amino acid residuesfrom the cation channel protein to form a truncated cation channelprotein, dissolving the truncated cation channel protein in a proteinsolution, such that the concentration of dissolved truncated channelprotein is about 5 to about 10 mg/ml, and mixing equal volumes ofprotein solution with reservoir mixture at 20° C. Preferably, thereservoir mixture comprises 200 mM CaCl₂, 100 mM Hepes, 48% PEG 400, pH7.5, and the protein solution comprises (150 mM KCl, 50 mM Tris, 2 mMDTT, pH 7.5).

Moreover, the present invention extends to a method of growing a crystalof a cation channel protein as described above, wherein a crystal can begrown comprising any type of cation channel protein. In particular, thepresent invention can be used to grow crystals of potassium channelproteins, sodium channel proteins, or calcium channel proteins, to nameonly a few.

Furthermore, the present invention extends to a method of growing acrystal of a cation channel protein, as described herein, wherein thecrystal comprises an amino acid sequence of:

-   -   residues 23 to 119 of SEQ ID NO:1 (Streptomyces lividans);    -   residues 61 to 119 of SEQ ID NO:2 (E. coli);    -   residues 61 to 119 of SEQ ID NO:3 (Clostridium acetobutylicum);    -   residues 61 to 119 of SEQ ID NO:4 (Drosophila melanogaster);    -   residues 61 to 119 of SEQ ID NO:5 (Homo sapiens);    -   residues 61 to 119 of SEQ ID NO:6 (Homo sapiens);    -   residues 61 to 119 of SEQ ID NO:7 (Paramecium tetraaurelia);    -   residues 61 to 119 of SEQ ID NO:8 (C. elegans);    -   residues 61 to 119 of SEQ ID NO:9 (Mus musculus);    -   residues 61 to 119 of SEQ ID NO:10 (Homo sapiens);    -   residues 61 to 119 of SEQ ID NO:11 (Arabidopsis thaliana);    -   residues 61 to 119 of SEQ ID NO:12 (Homo sapiens);    -   residues 61 to 119 of SEQ ID NO:13 (Rattus novegicus); or        residues 61 to 119 of SEQ ID NO:14 (Homo sapiens);        or conserved variants thereof.

Use of Crystal of a Cation Channel Protein in Assay Systems forScreening Drugs and Agents

In another embodiment, the present invention extends to a method ofusing a crystal of a cation channel protein, as described herein, in anassay system for screening drugs and other agents for their ability tomodulate the function of a cation channel protein, comprising the stepsof initially selecting a potential drug or agent by performing rationaldrug design with the three-dimensional structure determined for acrystal of the present invention, wherein the selecting is performed inconjunction with computer modeling. After potential drugs or agents havebeen selected, a cation channel protein is contacted with the potentialdrug or agent. If the drug or therapeutic agent has potential use formodulating the function of a cation channel protein, a change in thefunction of the cation channel after contact with the agent, relative tothe function of a similar cation channel protein not contacted with theagent, or the function of the same cation channel protein prior tocontact with the agent. Hence, the change in function is indicative ofthe ability of the drug or agent to modulate the function of a cationchannel protein.

Furthermore, the present invention extends to extends to a method ofusing a crystal of a cation channel protein as described herein, in anassay system for screening drugs and other agents for their ability tomodulate the function of a cation channel protein, wherein the crystalcomprises a Na⁺ channel protein, a K⁺ channel protein, or a Ca²⁺ channelprotein.

The present invention further extends to a method of using a crystal ofa cation channel protein in an assay for screening drugs or other agentsfor their ability to modulate the function of a cation channel protein,wherein the crystal of the cation channel protein comprises an aminoacid sequence of:

-   -   residues 23 to 119 of SEQ ID NO:1 (Streptomyces lividans);    -   residues 61 to 119 of SEQ ID NO:2 (E. coli);    -   residues 61 to 119 of SEQ ID NO:3 (Clostridium acetobutylicum);    -   residues 61 to 119 of SEQ ID NO:4 (Drosophila melanogaster);    -   residues 61 to 119 of SEQ ID NO:5 (Homo sapiens);    -   residues 61 to 119 of SEQ ID NO:6 (Homo sapiens);    -   residues 61 to 119 of SEQ ID NO:7 (Paramecium tetraaurelia);    -   residues 61 to 119 of SEQ ID NO:8 (C. elegans);    -   residues 61 to 119 of SEQ ID NO:9 (Mus musculus);    -   residues 61 to 119 of SEQ ID NO:10 (Homo sapiens);    -   residues 61 to 119 of SEQ ID NO:11 (Arabidopsis thaliana);    -   residues 61 to 1119 of SEQ ID NO:12 (Homo sapiens);    -   residues 61 to 119 of SEQ ID NO:13 (Rattus novegicus); or    -   residues 61 to 119 of SEQ ID NO:14 (Homo sapiens);        or conserved variants thereof.

In a preferred embodiment of a method of using a crystal of a cationchannel protein in an assay for screening drugs or other agents fortheir ability to modulate the function of a cation channel protein, thecrystal comprises a potassium channel protein, comprising amino acidresidues 23 to 119 of SEQ ID NO:1, a space grouping of C2, and a unitcell of dimensions of a=128.8 Å, b=68.9 Å, c=112.0 Å, and β=124.6°.

Moreover, it is important to note that a drug's or agent's ability tomodulate the function of a cation channel protein includes, but is notlimited to, increasing or decreasing the cation channel protein'spermeability to the specific cation relative the permeability of thesame or a similar not contacted with the drug or agent, or the samecation channel protein prior to contact with the drug or agent.

In a further embodiment, the present invention extends to a method ofusing a crystal of a cation channel protein, as set forth herein, in anassay system for screening drugs and other agents for their ability totreat conditions related to the function of cation channel proteins invivo, and particularly in abnormal cellular control processes related tothe functioning of cation channel protein. Such a method comprises theinitial step of selecting a potential drug or other agent by performingrational drug design with the three-dimensional structure determined fora crystal of the invention, wherein the selecting is performed inconjunction with computer modeling. After potential drugs or therapeuticagents are selected, a cation channel protein is contacted with thepotential drug or agent. If an interaction of the potential drug orother agent with the cation channel is detected, it is indicative of thepotential use of the drug or agent to treat conditions related thefunction of cation channel proteins in vivo. Examples of such conditionsinclude, but are not limited to, cardiac arrhythmia, diabetes mellitus,seizure disorder, asthma or hypertension, to name only a few.

Furthermore, a crystal of a cation channel protein used in the methodfor screening drugs or agents for their ability to interact with acation channel comprises an Na⁺ channel protein. K⁺ channel protein, orCa²⁺ channel protein. Hence, the method of the present invention can beused to screen drugs or agents capable of treating conditions related tothe function of such channels.

Moreover, the present invention extends to a crystal used in the methodfor screening drugs or agents for their ability to interact with acation channel protein comprising an amino acid sequence of:

-   -   residues 23 to 119 of SEQ ID NO:1 (Streptomyces lividans);    -   residues 61 to 119 of SEQ ID NO:2 (E. coli);    -   residues 61 to 119 of SEQ ID NO:3 (Clostridium acetobutylicum);    -   residues 61 to 119 of SEQ ID NO:4 (Drosophila melanogaster);    -   residues 61 to 119 of SEQ ID NO:5 (Homo sapiens);    -   residues 61 to 119 of SEQ ID NO:6 (Homo sapiens);    -   residues 61 to 119 of SEQ ID NO:7 (Paramecium tetraaurelia);    -   residues 61 to 119 of SEQ ID NO:8 (C. elegans);    -   residues 61 to 119 of SEQ ID NO:9 (Mus musculus);    -   residues 61 to 119 of SEQ ID NO:10 (Homo sapiens);    -   residues 61 to 119 of SEQ ID NO:11 (Arabidopsis thaliana);    -   residues 61 to 119 of SEQ ID NO:12 (Homo sapiens);    -   residues 61 to 119 of SEQ ID NO:13 (Rattus novegicus); or        residues 61 to 119 of SEQ ID NO:14 (Homo sapiens);        or conserved variants thereof.

In a preferred embodiment, a crystal used in a method for screeningdrugs or agents for their ability to interact with a cation channel,comprises amino acid residues 23 to 119 of SEQ ID NO:1, has a spacegrouping of C2, and a unit cell of dimensions of a=128.8 Å, b=68.9 Å,c=112.0 Å, and β=124.6°.

In yet another embodiment, the present invention extends to a method ofusing a crystal of a cation channel protein described herein, in anassay system for screening drugs and other agents for their ability topermeate through a cation channel protein, comprising an initial step ofselecting a potential drug or other agent by performing rational drugdesign with the three-dimensional structure determined for the crystal,wherein the selecting of the potential drug or agent is performed inconjunction with computer modeling. After a potential drug or agent hasbeen selected, a cation channel protein can be prepared for use in theassay. For example, preparing the cation channel protein can includeisolating the cation channel protein from the membrane of a cell, andthen inserting the cation channel protein into a membrane having a firstand second side which is impermeable to the potential drug or agent. Asa result, the cation channel protein traverses the membrane, such thatthe extracellular portion of the cation channel protein is located onthe first side of the membrane, and the intracellular portion of thecation channel protein is located on the second side of the membrane.The extracellular portion of the cation channel membrane can then becontacted with the potential drug or agent. The presence of the drug oragent in the second side of the membrane is indicative of the drug's oragent's potential to permeate the cation channel protein, and the drugor agent is selected based on its ability to permeate the cation channelprotein.

In addition, a crystal used in a method for screening drugs or agentsfor their ability to permeate a cation channel can comprise a Na⁺channel protein, a K⁺ protein channel, or a Ca²⁺ protein channel.

Furthermore, the present invention extends to the use of a crystal in anassay system for screening drugs and other agents for their ability topermeate through a cation channel protein, wherein the crystal comprisesan amino acid sequence of:

-   -   residues 23 to 119 of SEQ ID NO:1 (Streptomyces lividans);    -   residues 61 to 119 of SEQ ID NO:2 (E. coli);    -   residues 61 to 119 of SEQ ID NO:3 (Clostridium acetobutylicum);    -   residues 61 to 119 of SEQ ID NO:4 (Drosophila melanogaster);    -   residues 61 to 119 of SEQ ID NO:5 (Homo sapiens);    -   residues 61 to 119 of SEQ ID NO:6 (Homo sapiens);    -   residues 61 to 119 of SEQ ID NO:7 (Paramecium tetraaurelia);    -   residues 61 to 19 of SEQ ID NO:8 (C. elegans);    -   residues 61 to 119 of SEQ ID NO:9 (Mus musculus);    -   residues 61 to 119 of SEQ ID NO:10 (Homo sapiens);    -   residues 61 to 119 of SEQ ID NO:11 (Arabidopsis thaliana);    -   residues 61 to 119 of SEQ ID NO:12 (Homo sapiens);    -   residues 61 to 119 of SEQ ID NO:13 (Rattus novegicus); or        residues 61 to 119 of SEQ ID NO:14 (Homo sapiens);        or conserved variants thereof.

In a preferred embodiment, the crystal used in an assay system of thepresent invention for screening drugs and other agents for their abilityto permeate through a cation channel protein comprises amino acidresidues 23 to 119 of SEQ ID NO:1, has a space grouping of C2, and aunit cell of dimensions of a=128.8 Å, b=68.9 Å, c=112.0 Å, and β=124.6°.

In the assay systems disclosed herein, Once the three-dimensionalstructure of a crystal comprising a cation channel protein isdetermined, a potentia drugs and therapeutic agents which may interactwith a carrier channel protein, i.e. bind or modulate the functionthereof, or perhaps be able to permeate through such a protein can beexamined through the use of computer modeling using a docking programsuch as GRAM, DOCK, or AUTODOCK [Dunbrack et al., 1997, supra]. Thisprocedure can include computer fitting of potential drugs or agents to acation channel protein to ascertain how well the shape and the chemicalstructure of the potential drug or agent will complement or interactwith a cation channel protein. [Bugg et al., Scientific American,Dec.:92-98 (1993); West et al., TIPS, 16:67-74 (1995)]. Computerprograms can also be employed to estimate the attraction, repulsion, andsteric hindrance of a potential drug or agent to a cation channelprotein. Generally the tighter the fit, the lower the steric hindrances,and the greater the attractive forces, the more potent the potentialdrug or agent, since these properties are consistent with a tighterbinding, and are clearly indicative of an interaction with a cationchannel protein. Furthermore, the more specificity in the design of apotential drug the more likely that the drug will not interact as wellwith other proteins. This will minimize potential side-effects due tounwanted interactions with other proteins.

Furthermore, computer modeling programs based on the structure of acation channel protein in a crystal of the present invention, can beused to modify potential drugs or agents in order to identifypotentially more promising drugs. Such analysis has been shown to beeffective in the development of HIV protease inhibitors [Lam et al.,Science 263:380-384 (1994): Wlodawer et al., Ann. Rev. Biochem.62:543-585 (1993); Appelt, Perspectives in Drug Discovery and Design1:23-48 (1993); Erickson, Perspectives in Drug Discovery and Design1:109-128 (1993)]. Alternatively a potential drug or agent can beobtained by initially screening a random peptide library produced byrecombinant bacteriophage for example, [Scott and Smith, Science,249:386-390 (1990); Cwirla et al., Proc. Natl. Acad. Sci., 87:6378-6382(1990): Devlin et al., Science, 249:404-406 (1990)]. A peptide selectedin this manner would then be systematically modified by computermodeling programs in odrer to enhance its potential interaction with acation channel protein.

Moreover, through the use of the three-dimensional structure disclosedherein and computer modeling, a large number of these compounds can berapidly screened on the computer monitor screen, and a few likelycandidates can be determined without the laborious synthesis of untoldnumbers of compounds.

Once a potential drug or agent is identified, it can be either selectedfrom a library of chemicals as are commercially available from mostlarge chemical companies including Merck, GlaxoWelcome, Bristol MeyersSquib, Monsanto/Searle, Eli Lilly. Novartis and Pharmacia UpJohn,alternatively the potential drug or agent may be synthesized de novo.The de novo synthesis of one or even a relatively small group ofspecific compounds is reasonable in the art of drug design. Thepotential drug or agent can then be placed into an assay of the presentinvention to determine whether it binds with a cation channel protein.

When suitable potential drugs or agents are identified, a supplementalcrystal is grown which comprises a cation channel protein. Preferablythe crystal effectively diffracts X-rays for the determination of theatomic coordinates of the protein-ligand complex to a resolution ofgreater than 5.0 Angstroms, more preferably greater than 3.0 Angstroms,and even more preferably greater than 2.0 Angstroms. Thethree-dimensional structure of the supplemental crystal is determined byMolecular Replacement Analysis. Molecular replacement involves using aknown three-dimensional structure as a search model to determine thestructure of a closely related molecule or protein-ligand complex in anew crystal form. The measured X-ray diffraction properties of the newcrystal are compared with the search model structure to compute theposition and orientation of the protein in the new crystal. Computerprograms that can be used include: X-PLOR and AMORE [J. Navaza. ActaCrystallographics ASO, 157-163 (1994)]. Once the position andorientation are known an electron density map can be calculated usingthe search model to provide X-ray phases. Thereafter, the electrondensity is inspected for structural differences and the search model ismodified to conform to the new structure.

The present invention may be better understood by reference to thefollowing non-limiting Examples, which are provided as exemplary of theinvention. The following examples are presented in order to more fullyillustrate the preferred embodiments of the invention.

They should in no way be construed, however, as limiting the broad scopeof the invention.

EXAMPLE I

Potassium Channel Structure: Molecular Basis of K⁺ Conduction andSelectivity

The K⁺ channel from Streptomyces lividans is an integral membraneprotein with sequence similarity to all known K⁺ channels, particularlyin the pore region. X-ray analysis with data to 3.2 (reveals that fouridentical subunits create an inverted tepee, or cone, cradling theselectivity filter of the pore in its outer end. The narrow selectivityfilter is only 12 Å long, while the remainder of the pore is wider andlined with hydrophobic amino acids. A large, water-filled cavity andhelix dipoles are positioned so as to overcome electrostaticdestabilization of an ion in the pore at the center of the bilayer.Main-chain carbonyl oxygen atoms from the K⁺ channel signature sequenceline the selectivity filter, which is held open by structuralconstraints to coordinate K⁺ ions but not smaller Na⁺ ions. Theselectivity filter contains two K⁺ ions about 7.5 Å apart. Thisconfiguration promotes ion conduction by exploiting electrostaticrepulsive forces to overcome attractive forces between K⁺ ions and theselectivity filter. The architecture of the pore establishes thephysical principles underlying selective K⁺ conduction.

More particularly, potassium ions diffuse rapidly across cell membranesthrough proteins called K⁺ channels, which underlie many fundamentalbiological processes including electrical signaling in the nervoussystem. Potassium channels use diverse mechanisms of gating (theprocesses by which the pore opens and closes), but they all exhibit verysimilar ion permeability characteristics (1). All K⁺ channels show aselectivity sequence of K⁺ Rb⁺>Cs⁺, while permeability for the smallestalkali metal ions Na⁺ and Li⁺ is immeasurably low. Potassium is at leastten thousand times more permeant than Na⁺, a feature that is essentialto the function of K⁺ channels. Potassium channels also share aconstellation of permeability characteristics that is indicative of amulti-ion conduction mechanism. The flux of ions in one direction showshigh order coupling to flux in the opposite direction, and ionicmixtures result in anomalous conduction behavior (2). Because of theseproperties, K⁺ channels are classified as “long pore channels”, invokingthe notion that multiple ions queue inside a long, narrow pore insingle-file fashion. In addition, the pores of all K⁺ channels can beblocked by tetraethylammonium ions (3).

Molecular cloning and mutagenesis experiments have reinforced theconclusion that all K⁺ channels have essentially the same poreconstitution. Without exception, they contain a critical amino acidsequence that has been termed the K⁺ channel signature sequence.Mutation of these amino acids disrupts the channel's ability todiscriminate between K⁺ and Na⁺ ions (4).

Biophysicists have been tantalized for the past quarter century aboutchemical basis of the impressive fidelity with which the channeldistinguishes between K⁺ and Na⁺ ions, which are featureless spheres ofPauling radius 1.33 Å and 0.95 Å and the ability of K⁺ channels to beconcurrently so highly selective and exhibit a throughput rateapproaching the diffusion limit. The 10⁴ margin by which K⁺ is selectedover Na⁺ implies strong energetic interactions between K⁺ ions and thepore. And yet strong energetic interactions seem incongruent withthroughput rates up to 10⁸ ions per second.

Potassium Channel Architecture

Amino acid sequences show the relationship of the K⁺ channel fromStreptomyces lividans (kcsa K⁺ channel) (5) to other channels inbiology, including vertebrate and invertebrate voltage-dependent K⁺channels, vertebrate inward rectifier and Ca²⁺-activated K⁺ channels, K⁺channels from plants and bacteria, and cyclic nucleotide-gated cationchannels (FIG. 1). On the basis of hydrophobicity analysis, there aretwo closely related varieties of K⁺ channels, those containing twomembrane-spanning segments per subunit and those containing six. In allcases, the functional K⁺ channel protein is a tetramer (6), typically offour identical subunits (7). Subunits of the two membrane-spanningvariety appear to be shortened versions of their larger counterparts, asif they simply lack the first four membrane-spanning segments. Thoughthe kcsa K⁺ channel belongs to the two membrane-spanning set of K⁺channels, its amino acid sequence is actually closer to those ofeukaryotic six membrane-spanning K⁺ channels. In particular, itssequence in the pore region, located between the membrane-spanningstretches and containing the K⁺ channel signature sequence, is nearlyidentical to that found in the Drosophila (Shaker) and vertebratevoltage-gated K⁺ channels (FIG. 1). Moreover, through a study of thekcsa K⁺ channel interaction with eukaryotic K₊ channel toxins, asdescribed infra, it has been confirmed that the kcsa K⁺ pore structureis indeed very similar to that of eukaryotic K⁺ channels, and that itsstructure is maintained when it is removed from the membrane usingdetergent (8).

Furthermore, the kcsa K⁺ channel structure from residue position 23 to119 of SEQ ID NO:1 has been determined with X-ray crystallography (Table1). The cytoplasmic carboxyl terminus (residues 126 to 158 of SEQ IDNO:1) were removed in the preparation and the remaining residues weredisordered. The kcsa K⁺ channel crystals are radiation sensitive and thediffraction pattern is anisotropic, with reflections observed along thebest and worst directions at 2.5 Å and 3.5 Å Bragg spacings,respectively. By careful, data selection, anisotropy correction,introduction of heavy atom sites by site-directed mutagenesis, averagingand solvent flattening, an interpretable electron density map has beencalculated (FIG. 2, A-C). This map was without main chain breaks andshowed strong side chain density (FIG. 2C). The model was refined withdata to 3.2 Å (the data set was 93% complete to 3.2 Å with 67%completeness between 3.3 Å and 3.2 Å), maintaining highly restrainedstereochemistry and keeping tight noncrystallographic symmetry of cationchannel proteins, and that they all will have four inner helicesarranged like the poles of a tepee, four pore helices, and a selectivityfilter—tuned to select the appropriate cation—located close to theextracellular surface.

Surprisingly, this structure of the kcsa K⁺ channel is in excellentagreement with extensive functional and mutagenesis studies on Shakerand other eukaryotic K⁺ channels (FIG. 4).

The pore-region of K⁺ channels was first discovered with pore-blockingscorpion toxins (11). These inhibitors interact with amino acids (white)comprising the broad extracellular-facing entryway to the pore (12). Theimpermeant organic cation tetraethylammonium (TEA) blocks K⁺ channelsfrom both sides of the membrane at distinct sites (13). Amino acidsinteracting with externally and internally applied TEA are located justexternal to (yellow) and internal to (mustard) the structure formed bythe signature sequence amino acids (14, 15). Alteration of the signaturesequence amino acids (red main chain atoms) disrupts K⁺ selectivity (4).Amino acids close to the intracellular opening on the Shaker K⁺ channelmap to the inner helix on the kcsa K⁺ channel (16). Interestingly,exposure to the cytoplasm of the region above the inner helix bundle(pink side chains) requires an open voltage-dependent gate, whereas theregion at or below the bundle (green side chains) is exposed whether ornot the gate was open. The correlation between the transition zone forgate dependent exposure to the cytoplasm in the Shaker K⁺ channel andthe inner helix bundle in this structure has implications for mechanismsof gating in K⁺ channels.

General Properties of the Ion Conduction Pore

Both the intracellular and extracellular entryways are charged negativeby acidic amino acids (FIG. 5A, red), an effect that would raise thelocal concentration of cations while lowering the concentration ofanions. The overall length of the pore is about 45 Å and its diametervaries along its distance (FIG. 5B). From inside the cell (bottom) thepore begins as a tunnel about 18 Å in length (the internal pore) andthen opens into a wide cavity (about 10 Å across) near the middle of themembrane. A K⁺ ion could move throughout the internal pore and cavityand still remain mostly hydrated. In contrast, the selectivity filterseparating the cavity from the extracellular solution is so narrow thata K⁺ ion would have to shed its hydrating waters to enter. The chemicalcomposition of the wall lining the internal pore and cavity ispredominantly hydrophobic (FIG. 5A, yellow). The selectivity filter, onthe other hand, is lined exclusively by polar main chain atoms belongingto the signature sequence amino acids. The distinct mechanisms operatingin the cavity and internal pore versus the selectivity filter arediscussed below.

As explained above, potassium channel proteins exclude the smalleralkali metal cations Li⁺, (radius 0.60 Å) and Na⁺ (0.95 Å) but allowpermeation of the larger members of the series Rb⁻ (1.48 Å) and Cs⁺(1.69 Å). In fact Rb⁺ is nearly the perfect K⁺ (1.33 Å) analog as itssize and permeability characteristics are very similar to those of K⁺.Because they are more electron dense than K⁺, Rb⁺ and Cs⁺ allowvisualization of the locations of permeant ions in the pore. Bydifference electron density maps calculated with data from crystalstransferred into Rb⁺-containing (FIG. 6A) or Cs⁺-containing (FIG. 6B)solutions, multiple ions are well-defined in the pore. The selectivityfilter contains two ions (inner and outer ions) located at oppositeends, about 7.5 Å apart (center to center). In the Rb⁺ difference map,there actually are two partially separated peaks at the inner aspect ofthe selectivity filter. These peaks are too close to each other (2.6 Å)to represent two simultaneously occupied ion binding sites. AlthoughApplicant ise under no obligation to explain such peaks, and is not tobe bound by any explanations. Applicant merely postulates these peaksmay represent a single ion (on average) in rapid equilibrium betweenadjacent sites. The single inner ion peak in the Cs⁺ difference mapundoubtedly reflects the lower resolution at which the map wascalculated (to 5 Å for Cs⁺ versus 4.0 Å for Rb⁺) since the Rb⁺difference map, when calculated at the same lower resolution, also showsonly a single peak at the Cs⁺ position. The Rb⁺ positions correspond tostrong peaks (presumably K⁺ ions) in a high contour native electrondensity map (not shown). Thus, the selectivity filter may contain two K⁺ions. A third weaker peak is located below the selectivity fitter at thecenter of the large cavity in the Rb⁺ difference map (FIG. 6A, towerpeak) and in the Cs⁺ difference map at lower contour (not shown).Electron density at the cavity center is prominent in MIR maps evenprior to averaging (FIG. 6C, lower diffuse peak). The differenceelectron density maps show this to be related to the presence of one ormore poorly localized cations situated at least 4 Å away from theclosest protein groups.

The Cavity and Internal Pore

FIGS. 5B and 6 indicate that surprisingly, a 10 Å diameter cavity is inthe center of the channel protein with an ion in it. Electrostaticcalculations indicate that when an ion is moved alone a narrow porethrough a membrane it must cross an energy barrier that is maximum atthe membrane center (17). The electrostatic field emanating from acation polarizes its environment, bringing the negative ends of dipolescloser to it and thereby stabilizing it. At the bilayer center, thepolarizability of the surrounding medium is minimal and therefore theenergy of the cation is highest. Thus, simple electrostaticconsiderations allow an understanding of the functional significance ofthe cavity and its strategic location. The cavity will serve to overcomethe electrostatic destabilization resulting from the low dielectricbilayer by simply surrounding an ion with polarizable water. A secondfeature of the K⁺ channel structure will also stabilize a cation at thebilayer center. The four pore helices point directly at the center ofthe cavity (FIG. 3, A, B and D). The amino to carboxyl orientation ofthese helices will impose a negative electrostatic (cation attractive)potential via the helix dipole effect (18). The ends of the helices arerather far (about 8 Å) from the cavity center, but all four contributeto the effect. Therefore, two properties of the structure, the aqueouscavity and the oriented helices, help to solve a very fundamentalphysical problem in biology—how to lower the electrostatic barrierfacing a cation crossing a lipid bilayer. Thus, the diffuse electrondensity in the cavity center most likely reflects not an ion bindingsite, but rather a hydrated cation cloud (FIG. 7).

In summary, the inner pore and cavity lower electrostatic barrierswithout creating deep energy wells. The structural and chemical designof this part of the pore ensure a low resistance pathway from thecytoplasm to the selectivity filter, facilitating a high throughput.Functional experiments on K⁺ channels support this conclusion. When TEAfrom the cytoplasm migrates to its binding site at the top of thecavity, >50% of the physical distance across the membrane (FIG. 4 andFIG. 5), it traverses only about 20% of the transmembrane voltagedifference (15). Thus, 80% of the transmembrane voltage is imposedacross the relatively short selectivity filter. The rate limiting stepsfor a K⁺ ion traversing the channel are thereby limited to this shortdistance. In effect, the K⁺ channel has thinned the relevanttransmembrane diffusion distance to a mere 12 Å.

The Selectivity Filter

Construction of the atomic model for the K⁺ channel selectivity filterwas based on the experimental electron density map which showed acontinuous ridge of electron density attributable to the main chain, aswell as strong valine and tyrosine side chain density directed away fromthe pore (FIG. 8A). K⁺ ion positions defined by difference Fourieranalysis (FIG. 6 and FIG. 8A, yellow density) along with knowledge ofalkali metal cation coordination in small molecules were also used inthe construction. The side chain locations preclude their directparticipation in ion coordination, leaving this function to the mainchain atoms. The precise orientation of individual carbonyl oxygens cannot be discerned at the resolution of this X-ray analysis. AlthoughApplicant is under no obligation to explain the orientation ofindividual carbonyl atoms, and are not to be bound by such explanations.Applicant merely proposes they are directed inward to account for K⁺ ioncoordination (FIG. 8B). A single water molecule (the only one modeled inthe structure) located between the two K⁺ ions in the selectivity filterwas justified by the presence of a strong electron density peak in theexperimental map which was never associated with an ion peak in thedifference Fourier maps (19).

The structure of the selectivity filter exhibits two essential features.First, the use of main chain atoms creates a stack of sequential oxygenrings and thus affords numerous closely spaced sites of suitabledimensions for coordinating a dehydrated K⁺ ion. The K⁺ ion thus hasonly a very small distance to diffuse from one site to the next withinthe selectivity filter. The second important structural feature of theselectivity filter is the protein packing around it. The Val and Tyrside chains from the V-G-Y-G sequence point away from the pore and makespecific interactions with amino acids from the tilted pore helix. Incollusion with the pore helix Trp residues, the four Tyr side chainsform a massive sheet of aromatic amino acids, twelve in total, that ispositioned like a cuff around the selectivity filter (FIG. 8C). Thehydrogen bonding, for example between the Tyr hydroxyls and Trpnitrogens, and the extensive van der Waals contacts within the sheet,offer the immediate impression that this structure behaves like a layerof springs stretched radially outward to hold the pore open at itsproper diameter.

Applicant postulates, although under no obligation to do so, and not tobe bound thereby, that when an ion enters the selectivity filter itevidently dehydrates (nearly completely). To compensate for theenergetic cost of dehydration, the carbonyl oxygen atoms must take theplace of the water oxygen atoms. That is, they must come in very closecontact with the ion and act like surrogate water (20, 21). Thestructure reveals that the selectivity filter is being held open as ifto prevent it from accommodating a Na⁺ ion with its smaller radius.

Therefore, Applicant postulates that a K⁺ ion fits in the filter justright, so that the energetic costs and gains are well balanced. Sodiumon the other hand is too small. The structure of the selectivity filterwith its molecular springs holding it open prevents the carbonyl oxygenatoms from approaching close enough to compensate for the cost ofdehydration of a Na⁺ ion.

This analysis shows that the selectivity filter contains two K⁺ ions inthe presence of about 150 mM K⁻ (FIG. 6 and FIG. 8). The ions arelocated at opposite ends of the selectivity filter, separated by about7.5 Å. That is roughly the average distance between K⁺ ions in a 4 MolarKCl solution, and in the selectivity filter there are no intervening Cl⁻anions to balance the charge. Although under no obligation to explainsuch results, and without intending to be bound by any explanation,Applicant postulates, that the selectivity filter attracts andconcentrates K⁺ ions. The structure implies that a single K⁺ ion wouldbe held very tightly, but that the presence of two K⁺ ions results inmutual repulsion, hence their locations near opposite ends of theselectivity filter. Thus, when a second ion enters, the attractive forcebetween a K⁺ ion and the selectivity filter becomes perfectly balancedby the repulsive force between ions, and this is what allows conductionto occur. This picture accounts for both a strong interaction between K⁺ions and the selectivity filter and a high throughput mediated byelectrostatic repulsion. On the basis of functional measurements, thesame concept of destabilization by multiple ion occupancy has beenproposed for Ca²⁺ channels (22) and for K⁺ channels (23) and perhaps isa general property of all selective ion channels.

Experimental Procedures

Cloning and Expression of the kcsa Gene

The kcsa gene was subcloned into pQE60 (Qiagen) vector and expressed inE. coli XL-1 Blue cells upon induction with 1-β-D-thiogalactopyranoside.The carboxy-terminal histidine tagged protein was extracted byhomogenization and solubilization in 40 mM decylmaltoside (Antrace). Thekcsa K⁺ channel was purified on a cobalt affinity column. Thirty-fivecarboxyl terminal amino acids were cleaved by chymotripsin proteolysis.The truncated channel was purified to homogeneity by gel filtration andthe detergent exchanged in a final dialysis step against 5 mMN,N,-dimethyldodecylamine-N-oxide (LDAO). Crystals were grown at 20° C.by using the sitting drop method by mixing equal volumes of asolubilizing solution with reservoir mixture. Through the entirepreparation, the channel protein was maintained in solutions containing150 mM KCl. For definition of K⁺ sites, crystals were transferred intosolutions where 150 mM KCl was replaced by 150 mM RbCl or 150 mM CsCl.

X-Ray Crystallography

Crystals (space group C2: α=128.8 Å, b=68.9 Å, c=112.0 Å, β=124.6° wereflash-frozen by transferring directly from the crystal mother liquor toa stream of boiled-off nitrogen (24). Since crystals of the mutant L90Cdiffracted significantly better than wild type protein crystals, theformer were used for native data collection. Data were collected frommultiple crystals and six sets were selected and merged to form thenative data set used for structure determination. Mercury derivativeswere obtained by direct addition of methyl mercury to thecrystallization solution of cysteine mutant crystals. MALDI-TOF massspectrometry confirmed 60-90% derivatization of crystals prior to datacollection. All data were collected at Cornell High Energy SynchrotronSource (CHESS), station A1, using the Princeton 2K CCD (25). Data wereprocessed with DENZO and SCALEPACK (26) and the CCP4 package (27). Heavyatom positions were determined with SHELX-97 (28) and cross-differenceFourier analysis. These positions confirmed the four-foldnoncrystallographic symmetry observed in the self-rotation Pattersonfunction and allowed the determination of initial orientation matrices.An initial model (90% complete) was built into a solvent flattened (64%solvent content), four-fold averaged electron density map using theprogram O (29). The tracing of the model was facilitated by the use ofthe mercury atom positions as residue markers. L86C was used solely forthis purpose. After torsional refinement (with strict four-foldnoncrystallographic symmetry constraints) using XPLOR 3.851 (30), thismodel was used in the anisotropic scaling (sharpening (31)) of thenative data with XPLOR. The structure factor sigma values were alsorescaled appropriately and the corrected data were used for allsubsequent procedures. Four-fold averaging, solvent flattening and phaseextension were applied in DM (32), resulting in a marked improvement ofthe electron density that allowed correction of the model and thebuilding of additional residues. Refinement consisted of rounds ofpositional (in the initial tages phase information was also included asa restraint) and grouped B-factor refinement in XPLOR. Four-foldnoncrystallographic symmetry was highly restrained with the forceconstant for positional restraints set as 1000 kcal/mol/Å². The diffuseion cloud described in the text was initially modeled as one or more K⁺ions and several water molecules, however the results wereunsatisfactory. Therefore, this and other strong unmodeled densitypresent in solvent flattened maps (no averaging included) was Fourierback-transformed, scaled and included in the refinement procedure, aspartial structure factors. The final model includes amino acids 23 to119 of each chain. The following residues were truncated: Arg27 to Cβ,Ile60, to Cγ, Arg64 to Cβ, Glu71 to Bβ and Arg117 to Nε. Thestereochemistry is strongly restrained, with no outliers on theRamachandran plot. The high B-factor values reflect the intensity decayof the data beyond 4 Å.

SUMMARY

Without intending to be bound by such proposals, and with no obligationto explain these results, Applicant proposes the following principlesunderlying the structure and operation of K⁺ channels. (i) The porestructure defines an inverted tepee architecture with the selectivityfilter held at its wide end. This architecture also describes the poreof cyclic nucleotide-gated channels and probably Na⁺ and Ca²⁺ channelsas well. (ii) The narrow selectivity filter is only 12 Å long, whilesurprisingly, the remainder of the pore is wider and has a relativelyinert hydrophobic lining. These structural and chemical properties favora high K⁺ throughput by minimizing the distance over which K⁺ interactsstrongly with the channel. (iii) A large water-filled cavity and helixdipoles help to overcome the high electrostatic energy barrier facing acation in the low dielectric membrane center. (iv) The K⁺ selectivityfilter is lined by carbonyl oxygen atoms providing multiple closelyspaced sites. The filter is constrained in an optimal geometry so that adehydrated K⁺ ion fits with proper coordination while the Na⁺ ion is toosmall. (v) Two K⁺ ions at close proximity in the selectivity filterrepel each other. The repulsion overcomes the otherwise stronginteraction between ion and protein and allows rapid conduction in thesetting of high selectivity. TABLE 1 Summary of data collection andrefinement statistics. Data Collection and Phasing: ResolutionCompleteness Phasing Dataset (Å) Redudancy Overall/outer Rmerge # Power¶ R-Cullis + L90C-a 15.0-3.7 3.5 91.3/93.3% 0.071 1.61 0.70 L90C-b15.0-3.7 7.0 91.5/94.1% 0.083 1.87 0.50 V93C 15.0-3.7 4.1 98.3/99.1%0.075 1.35 0.63 A32C 15.0-4.0 2.3 84.1/83.8% 0.076 1.45 0.66 A29C15.0-5.0 2.7 73.9/74.0% 0.063 1.03 0.85 A42C 15.0-6.5 2.0 90.7/90.3%0.057 0.97 0.81 L86C 30.0-6.0 2.3 58.7/58.9% 0.057 — —

% of measured I/σI data with I/σI>2 Native 30.0-3.2 6.1 93.3% 0.086 15.879 Outer Shell  3.3-3.2 2.3 66.6% 0.286 3.9 50 Anisotropic correction:Average F.O.M* Average F.O.M* (30.0-3.2 Å) (3.4-3.2 Å) Before Sharpening∂ 0.76 0.55 After sharpening ∂ 0.83 0.64 Refinement: Root-mean-squaredeviation of Resolution 10.0-3.2 Å bond angles: 1.096° R-cryst. &: 28.0%bond lengths 0.005 Å R-free &: 29.0% ncs related atoms: 0.006 Å No. ofreflections with|F|/σ|F| > 2: 12054 related atoms: 10 Å² No. of proteinatoms: 710 per subunit B-factor for non- No. of ligand atoms: 1 water, 3K⁺atoms bonded atoms: 36 Å² Mean B-factor for 90 Å² side-chain atoms:Mean B-factor for 110 Å² side-chain atoms:# Rmerge = ΣΣI-Ij/ΣI.:¶ Phasing power = <|Fh|>/<E>:-R-Cullis = Σ||Fph ± Fp|-|Fhc||/Σ|Fph ± Fp|. only for centric data: &R-cryst. = Σ|Fp-Fp(calc)|/Σ|Fp|. r-free the same for R-cryst., butcalculated on 10% of data selected in thin resolution shells andexcluded from refinement:*F.O.M.: figure of merit; σ in both cases four-fold averaging andsolvent flattening were applied:Ij is the observed intensity.I is the average intensity.Fh is the root-mean-square heavy-atom structure factor.E is the lack of closure error,Fph is the structure factor for the derivative,Fp is the structure factor for the native.Fhc is the calculated structure factor for the heavy-atom,Fp(calc) is the calculated native structure value.

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18. D. Sali, M. Bycroft, A R. Fersht, Nature 335, 740 (1988); J. Aqvist,H. Luecke, F. A. Quiocho, A. Warshel, Proc. Natl. Acad. Sci. U.S.A. 88,2026 (1991); D. J. Lockhart and P. S. Kim, Science 257, 947 (1992); D.J. Lockhart and P. S. Kim, Science 260, 198 (1993).

-   19. The temperature factors for Val76 and Gly77 main chain atoms    (but not side chain atoms) refined to higher values than for    neighboring atoms. This result is explicable based on the difference    Fourier analysis showing alternative positions of the inner K⁺ ion    in the selectivity filter and therefore, by inference, alternative    conformations of the coordinating main chain atoms, depending on the    location of the K⁺ ion.-   20. F. Bezanilla and C. M. Armstrong, J. Gen. Physiol. 60, 588    (1972).-   21. Hille, ibid. 61, 669 (1973).-   22. W. Almers and E. W. McCleskey, J. Physiol, (Lond.) 353, 585    (1984); P. Hess and R. W. Tsien, Nature 309, 453 (1984).-   23. J. Neyton and C. Miller, J. Gen. Physiol. 92, 569 (1988).-   24. The kcsa gene was subcloned into pQE60 (Qiagen) vector and    expressed in E. coli XL-1 Blue cells upon induction with    1-(-D-thiogalactopyranoside. The carboxyl-terminal histidine tagged    protein was extracted by homogenization and solubilization in 40 mM    decylmaltoside (Anatrace). The kcsa channel was purified on a cobalt    affinity column. Thirty-five carboxyl terminal amino acids were    cleaved by chymotrypsin proteolysis. The truncated channel was    purified to homogeneity by gel filtration and the detergent    exchanged in a final dialysis step against 5 mM    N,N-dimethyldodecylamine-N-oxide (LDAO). Crystals were grown at 20    (C by using the sitting drop method by mixing equal volumes of    protein solution (5-10 mg/ml. 150 mM KCl. 50 mM Tris pH 7.5, 2 mM    DTT) with reservoir mixture (200 mM CaCl₂, 100 mM Hepes pH 7.5 and    48% PEG 400). Through the entire preparation the channel protein was    maintained in solutions containing 150 mM KCl. For definition of K⁺    sites, crystals were transferred into solutions where 150 mM KCl was    replaced by 150 mM RbCl or 150 mM CsCl.-   25. M. W. Tate et al., J. Appl. Cryst. 28, 196 (1995); D. J. Thiel,    et al., Rev. Sci. Instrum. 67, 1 (1996).-   26. Z. Otwinowski, in Data Collection and Processing, L. Sawyer    and S. Bailey, Eds. (Science and Engineering Research Council    Daresbury Laboratory, Daresbury, UK, 1993), pp. 56-62.-   27. Collaborative Computational Project 4 (CCP4), Acta Cryst. DS50,    760(1994).-   28. G. M. Sheldrick, Acta Cryst. 46, 467 (1990).-   29. T. A. Jones. J. Y. Zou. J. Y. Cowan, M. Kjeldgaard, ibid. A47,    110 (1991).-   30. A. T. Brunger. X-Plor (Version 3.851) Manual (New Haven, Conn.:    The Howard Hughes Medical Institute and Department of Molecular    Biophysics and Biochemistry, Yale University).-   31. S. J. Gamblin, D. W. Rodgers, T. Stehle, Proceedings of the CCP4    Study weekend, Daresbury Laboratory, (1996) pp. 163-169.-   32. K. Y. J. Zhang and P. Main, Acta Cryst. A46, 377.-   33. P. J. Kraulis, J. Appl. Cryst. 24, 946 (1991).-   34. O. S. Smart. J. G. Neduvelil, X. Wang, B. A. Wallace, M. P.    Sansom, J. Mol. Graphics 14, 354 (1996).

EXAMPLE II Structural Conservation in Prokaryotic and Eukaryotic K⁻Channels Revealed by Scorpion Toxins

Scorpion toxins inhibit ion conduction through K⁺ channels by occludingthe pore at their extracellular opening. A single toxin protein bindsvery specifically to a single K⁺ channel to cause inhibition. The toxinsare 35 to 40 amino acids in length and have a characteristic fold thatis held rigidly by three disulfide bridges (1). They are active siteinhibitors, because when they bind to the channel they interactenergetically with K⁺ ions in the pore (2-4). The intimate interactionbetween these inhibitors and the pore of K⁺ channels has been exploitedto gain insights into the structure and function of K⁺ channels.

Studies employing site-directed mutagenesis of the Shaker K⁺ channelhave mapped the scorpion toxin binding site to regions corresponding tothe extracellular entryway of the kcsa K⁺ channel (4-9). Although the K⁺channel selectivity filter amino acids are highly conserved, theresidues lining the entryway are quite variable. As if to mirror theamino acid variation at the binding site, the toxins are also highlyvariable in their amino acid composition. A given scorpion venom is averitable library of toxins, apparently ensuring that a scorpion willinhibit a large fraction of K⁺ channel types in its victim. Studies onthe specificity of toxin-channel interactions have led to the followingunderstanding. The extracellular entryway to the K⁺ channel isrelatively conserved in its three-dimensional structure but the preciseamino acid composition is not conserved. The scorpion toxins have ashape, dictated by their conserved fold, that enables them to fit snuglyinto the entryway, but the affinity of a given toxin-channel pairdepends on the residue match (or mismatch) on both interaction surfaces.

A study of the interaction between the kcsa K⁺ channel (5) and thescorpion toxin agitoxin2 has been undertaken (10). By producing, throughmutagenesis, a competent toxin binding site, it is shown that the kcsaK⁺ channel pore structure and extracellular entryway is very similar tothat of eukaryotic voltage-gated K⁺ channels such as the Shaker K⁺channel from Drosophila and the vertebrate voltage-gated K⁺ channels,and that mutated potassium channel proteins of prokaryotic organismsmimic the physiological functions and chemical properties of eukaryoticcation binding proteins. By combining functional data collected on thetoxin-channel interaction with the structures of both proteins Applicantproposes, without intending to be bound by such proposals, ahighly-restrained model of the complex structure.

Experimental Procedures

Three mutations (Q58A, T61 S, R64D) were introduced into the kcsa K⁺channel gene to modify its pore region sequence using PCR mutagenesisand confirmed by DNA sequencing. The gene also contained a mutation atthe second residue (P2A) to introduce an ncol restriction endonucleasesite and it was lacking the last two carboxyl terminal residues (bothArg) to avoid proteolysis during the protein preparation. This gene wascloned into the pQE60 vector for expression with a carboxyl terminalthrombin and hexahistidine fusion. Channel protein was expressed in XL-1Blue strain of E. coli (Stratagene) by induction withl-β-D-thiogalactopyranoside at a concentration of 1.0 mM. Three hoursfollowing induction bacteria were sonicated in 50 mM Tris buffer (7.5),100 mM KCl. 10 mM Mg₂SO₄, 25 mg DNAse 1, 250 mM sucrose, in addition topepstatin, leupeptin, and PMSF. The channel was extracted in the samesolution containg 40 mM decylmaltoside (Anatrace) at room temperature.Following centrifugation the supernatant was bound to cobalt resin(Talon) at a protein to resin ratio that will saturate the resin. Theresin was washed, and detergent concentration was lowered to 10.0 mM.One mL columns were prepared. The control resin (no channel) was handledin the same manner. The resin preparation was the same for massspectrometry and binding studies.

Forty mg of Leiurus quinquestriatus hebraeus venom, (Alomone Labs) wassuspended in buffer identical to that of the channel (10.0 mMdeclymaltoside) and applied to the column. After washing, channel waseluted with 1.0 M imidazole in the same buffer.

Wild type and mutant agitoxin2 were prepared (10). TritiatedN-ethylmaleimide (NEN Life Sciences) was conjugated to agitoxin2 D20C(14). Binding, was performed in a 300 μL volume containing 50 mM Tris(7.5), 100 mM KCl, 10 mM declymaltoside, and 0.3 μL of cobalt resinsaturated with the mutant kcsa K⁺ channel for 30 minutes at roomtemperature. Following brief centrifugation the supernatant was removed,resin was applied to a filter, rinsed briefly with ice cold buffer, andthen counted in a scintillation counter. All binding measurements weremade with a paired control containing a saturating concentration (200times K_(D)) of unlabeled wild type agitoxin2 to determine nonspecificbinding. The competition assay was carried out under the sameconditions. Labeled Agitoxin2 at 0.06 μM was always present andunlabeled toxin was added to compete with bound labeled toxin.

Discussion

Guided by knowledge of the toxin receptor on the Shaker K⁺ channel, setforth in SEQ ID NO:4, three point mutations were introduced into thekcsa K⁺ channel (SEQ ID NO:1) that should render it sensitive toscorpion toxins (FIG. 9). The amino acid sequence of the mutated kcsa K⁺is set forth in SEQ ID NO:16. Amino acids 61 and 64 of SEQ ID NO:1 werechanged to their Shaker K⁺ channel counterpart, and 58 was changed toalanine since a small side chain at this latter position favors binding(4, 7). The mutant kcsa K⁺ channel protein was expressed in Escherichiacoli, extracted from the membrane with the detergent decylmaltoside, andbound to cobalt resin through a carboxyl terminal hexahistidine tag(11). A 1 mL column, prepared with the K⁺ channel-containing resin, wasused to screen the venom of the Middle East scorpion Leiurusquinquestriatus hebraeus, the source of numerous well-characterized ionchannel toxins. Forty milligrams of venom was added to the column andafter washing, the K⁺ channel protein was eluted with an imidazolesolution (12). The eluate was analyzed with MALDI-TOF mass spectrometry,focusing on the low mass range appropriate for scorpion toxins (about4000 Da). The K⁺ channel column resulted in a dramatic enhancement ofspecific peaks (FIG. 10, A-C). Three of these peaks corresponded in massto the known K⁺ channel toxins agitoxin2, charybdotoxin, and Lq2 (FIG.10, C and D). A fourth peak (FIG. 10C, asterisk) may represent a noveltoxin, which is currently under study. However, Applicant is under noobligation to explain this peak, and is not bound by any theories setforth herein regarding this peak. The peak corresponding to chlorotoxin,a reported chloride channel inhibitor (13), did not bind and provides anindication of the degree to which the K⁺ channel toxins are purified bythe mutant kcsa K⁺ channel column (FIG. 10, A and C).

Further quantitative analysis was carried out with agitoxin2.Radiolabeled agitoxin2 was prepared by producing the mutation D20C inthe toxin (located far from its channel binding surface) and conjugatingit with tritiated N-ethylmaleimide (14). A filter assay showed thatlabeled agitoxin2 binds to the mutant kcsa K⁺ channel with anequilibrium dissociation constant, K_(D), of about 0.6 mM (FIG. 11A). Incontrast, no binding to the wild type channel could be detected (notshown). The total capacity of resin saturated with mutant channelprotein, based on the specific activity of radiolabeled toxin and theknown 1:1 stoichiometry (one toxin per tetrameric channel), is nearly 50pmoles of channel per μL of resin. This value approximates the expectedcapacity of the resin and therefore implies that all of the channel inthe preparation must have a correct conformation.

Amino acids in a well-defined region of agitoxin2 form its functionalinteraction surface, as determined by the effects of alaninesubstitution on binding to the Shaker K⁺ channel [FIG. 11C (4, 8)].Mutation of Lys 27 and Asn 30 had the largest destabilizing effects. Itis noteworthy that Lys 27 is conserved in all members of this toxinfamily because its side chain apparently plugs the pore of K⁺ channels(3). To confirm that agitoxin2 uses the same amino acids to interactwith the mutant kcsa K⁺ channel, the effects of the K27A and N30A toxinmutations with a competition binding assay were studied (FIG. 11B).These mutations decreased the affinity for the toxin significantly(130-fold and 45-fold, respectively), as anticipated from the Shaker K⁺channel studies. In contrast, the D20C mutation (predicted to be on theback side of the toxin), even with a bulky N-ethylmaleimide adduct, didnot influence affinity (FIG. 11. A and B). These results indicate thatagitoxin2 binds in the same manner to both the mutant prokaryotic kcsaK⁺ channel protein and the eukaryotic Shaker K⁺ channel protein. Theaffinity for the Shaker K⁺ channel is considerably higher (K_(D) ⁺1 nM),but only three amino acids have been mutated in the prokaryotic cationchannel protein to mimic the site on the Shaker K⁺ channel (FIG. 9).

These results demonstrate that the overall structure of the agitoxin2receptor site is very similar on both the kcsa and Shaker K⁺ channels.This conclusion justifies the use of energetic data borrowed from ShakerK⁺ channel studies to assist in the docking of agitoxin2 onto the kcsaK⁺ channel structure. Thermodynamic mutant cycle analysis has allowedthe identification of numerous energetically coupled residue pairs onthe interface [pairs of residues that are related by the fact thatmutating one influences the effect (on equilibrium binding) of mutatingthe other (8)]. The four best defined of these residue pairs aredisplayed in matched colors on the kcsa K⁺ channel and agitoxin2surfaces (FIG. 12 A). The three off-center residue pairs (blue, green,yellow) have the strongest mutant cycle coupling energies [>3 kT (4,8)]. The central residue pair (red) is coupled by 1.7 kT and independentinformation places Lys 27 (red residue on agitoxin2, FIG. 11 A) over thepore (3, 4). More visual inspection suggests a unique orientation forthe toxin on the channel (FIG. 12 B). If the toxin is placed with itsfunctionally defined interaction surface face-down in the groove formedby the turrets (5), with Lys 27 at the center, the colors match well inthree dimensions. The toxin seems to fit perfectly into the vestibule ofa K⁺ channel. The four-fold symmetry of the K⁺ channel provides fourstatistically distinguishable but energetically identical orientationsavailable for a toxin to bind [(FIG. 12 A) (15)].

In summary, through a combination of structural and functional data, itis shown that prokaryotic channel proteins can be mutated to mimic thephysiological functions and chemical properties of eukaryotic channelproteins. Furthermore, disclosed herein is a view of a K⁺ channel incomplex with a neurotoxin from scorpion venom. The kcsa K⁺ channel isstructurally very similar to eukaryotic K⁺ channels. This unexpectedstructural conservation, determined through application of techniquesdeveloped here, can be exploited to advance our understanding of K⁺channel pharmacology, and prepare mutant prokaryotic channel proteinsthat can be used to screen potential drugs or agents that may interactwith eukaryotic cation channel proteins in vivo, and treat conditionsrelated to the function of proteins.

REFERENCES

The following references, along with other relevant information wascited in Example II, and set forth below. All references cited inExample II are hereby incorporated by reference in their entirety.

-   1. M. L. Garcia et al., J. Bioenerg. Biomemb. 23, 615 (1991); C.    Miller, Neuron 15, 5 (1995).-   2. R. MacKinnon and C. Miller, J. Gen. Physiol. 91, 335 (1988); K.    M.-   3. C. S. Park and C. Miller, Neuron 9, 307 (1992).-   4. R. Ranganathan. J. H. Lewis. R. MacKinnon. Neuron 16, 131 (1996).-   5. D. A. Doyle et al., Science xxxx (1998).-   6. R. MacKinnon and C. Miller, Science 245, 1382 (1989); R.    MacKinnon, L. Heginbotham, T. Abramson, Neuron 5, 767 (1990); M.    Stocker and C. Miller, Proc. Natl. Acad. Sci. USA 91, 9509    (1994); D. Naranjo and C. Miller, Neuron 16, 123 (1996).-   7. S. Goldstein, D. J. Pheasant, C. Miller, Neuron 12, 1377 (1994).-   8. P. Hidalgo and R. MacKinnon, Science 268, 307 (1995).-   9. A. Gross and R. MacKinnon, Neuron 16, 399 (1996).-   10. M. L. Garcia et al., Biochemistry 33, 6834 (1994).-   11. Three mutations (Q58A, T61S, R64D) were introduced into the kcsa    K⁺ channel gene to modify its pore region sequence using PCR    mutagenesis and confirmed by DNA sequencing. The gene also contained    a mutation at the second residue (P2A) to introduce an ncol    restriction endonuclease site and it was lacking the last two    carboxyl terminal residues. This gene was cloned into the pQE60    vector for expression with a carboxyl terminal thrombin and    hexahistidine fusion. Channel protein was expressed in XL-1 Blue    strain of E. coli (Stratagene) by induction with    1-β-D-thiogalactopyranoside at a concentration of 1.0 mM. Three    hours following induction bacteria were sonicated in 50 mM Tris    buffer (7.5), 100 mM KCl, 10 mM Mg₂SO₄, 25 mg DNAse 1, 250 mM    sucrose, in addition to pepstatin, leupeptin, and PMSF. The channel    was extracted in the same solution containg 40 mM decylmaltoside    (Anatrace) at room temperature. Following centrifugation the    supernatant was bound to cobalt resin (Talon) at a protein to resin    ratio that will saturate the resin. The resin was washed, and    detergent concentration was lowered to 10.0 mM. One mL columns were    prepared. The control resin (no channel) was handled in the same    manner. The resin preparation was the same for mass spectrometry and    binding studies.-   12. Forty mg of Leiurus quinquestriatus hebraeus venom (Alomone    Labs) was suspended in buffer identical to that of the channel (10.0    mM declymaltoside) and applied to the column. After washing, channel    was eluted with 1.0 M imidazole in the same buffer, 13. J. A.    Debin, J. E. Maggio, G. R. Strichartz, Am. J. Physiol. Soc. 264,    C369 (1993); G. Lippens, J. Najib, S. J. Wodak, A. Tartar,    Biochemistry 34, 13 (1995).-   14. S. K. Aggarwal and R. MacKinnon, Neuron 16, 1169 (1996).-   15. R. MacKinnon, Nature 350, 232 (1991).-   16. S. L. Cohen and B. T. Chait, Anal. Chem. 68, 31 (1996).-   17. Wild type and mutant agitoxin2 were prepared (10). Tritiated    N-ethylmaleimide (NEN Life Sciences) was conjugated to agitoxin2    D20C (14). Binding was performed in a 300 μL volume containing 50 mM    Tris (7.5), 100 mM KCl, 10 mM declymaltoside, and 0.3 μL of cobalt    resin saturated with the mutant kcsa K⁺ channel for 30 minutes at    room temperature. Following brief centrifugation the supernatant was    removed, resin was applied to a filter, rinsed briefly with ice cold    buffer, and then counted in a scintillation counter. All binding    measurements were made with a paired control containing a saturating    concentration (200 times K_(d)) of unlabeled wild type agitoxin2 to    determine nonspecific binding. The competition assay was carried out    under the same conditions. Labeled Agitoxin2 at 0.06 μM was always    present and unlabeled toxin was added to compete with bound labeled    toxin.-   18. A. M. Krezel et al., Prot. Sci. 4 1478 (1995).-   19. A. Nicholls. K. A. Sharp, B. Honig, Proteins 11, 281 (1991).-   20. T. A. Jones, J. Y. Zou, J. Y. Cowan, M. Kjeldgaard, Acta Cryst.    A47, 110 (1991).

The present invention is not to be limited in scope by the specificembodiments describe herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

It is further to be understood that all base sizes or amino acid sizes,and all molecular weight or molecular mass values, given for nucleicacids or polypeptides are approximate, and are provided for description.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties.

1. A method of using a functional cation channel protein in an assay forscreening potential drugs or agents which interact with the cationchannel protein, the method comprising the steps of: a) providing afunctional cation channel protein; b) conjugating the functional cationchannel protein to a solid phase resin; c) contacting the potential drugor agent to the functional cation channel protein conjugated to thesolid phase resin; d) removing the functional cation channel proteinfrom the solid phase resin; and e) determining whether the potentialdrug or agent is bound to the cation channel protein.
 2. The method ofclaim 1, wherein the providing step comprises: a) expressing an isolatednucleic acid molecule encoding the cation channel protein in aunicellular host such that the cation channel protein is present in thecell membrane of the unicellular host; b) lysing the unicellular host ina solubilizing solution so that the cation 4 channel protein issolubilized in the solution; and c) extracting the cation channelprotein from the solublizing solution with a detergent.
 3. The method ofclaim 2, wherein lysing the unicellular host in a solubilizing solutioncomprises sonicating the unicellular host in a solution comprising 50 mMTris buffer, 100 mM KCl, 10 mM MgSO₄, 25 mg DNAse 1, 250 mM sucrose,pepstatin, leupeptin, and PMSF, pH 7.5.
 4. The method of claim 2,wherein the detergent comprises 40 mM decylmaltoside.
 5. The method ofclaim 1, wherein the conjugating step comprises binding the cationchannel protein to a cobalt resin at protein to resin ratio that allowsfor saturation of the resin with the cation channel protein.
 6. Themethod of claim 1, wherein the removing step comprises contacting thecation channel protein conjugated to the solid phase resin to animidazole solution.
 7. The method of claim 1, wherein the isolatednucleic acid molecule encoding the cation channel protein comprises aDNA sequence of SEQ ID NO:17, or degenerate variants thereof, or anisolated nucleic acid molecule hybridizable under standard hybridizationconditions to an isolated nucleic acid molecule comprising a DNAsequence of SEQ ID NO:17, or degenerate variants thereof.
 8. The methodof claim 1, wherein the potential drug or agent is a member of alibarary of compounds, and the contacting step comprises contacting thelibrary of compounds to the functional cation channel protein conjugatedto the solid phase resin.
 9. The method of claim 8, wherein the libraryof compounds comprises a mixture of compounds or a combinatoriallibrary.
 10. The method of claim 9, wherein the combinatorial librarycomprises a phage display library, or a synthetic peptide library.
 11. Aprokaryotic cation channel protein mutated to mimic a functionaleukaryotic cation channel protein.
 12. The prokaryotic cation channelprotein of claim 11, selected from the group consisting of a potassiumchannel protein, a sodium channel protein, or a calcium channel protein.13. The prokaryotic cation channel protein of claim 11, endogenouslyproduced in a prokaryotic organism selected from the group consisting ofE. coli, Streptomyces lividans, Clostridium acetobutylicum, orStaphylcoccus aureus.
 14. The prokaryotic cation channel protein ofclaim 11, comprising an amino acid sequence of SEQ ID Nos: 1, 2, 3, or7.
 15. The prokaryotic cation channel protein of claim 11, wherein saidprokaryotic cation channel protein is a potassium channel protein fromStreptomyces lividans.
 16. The prokaryotic cation channel of claim 15,encoded by a nucleic acid comprising a DNA sequence of SEQ ID NO:17, ordegenerate variants thereof.
 17. The prokaryotic cation channel proteinof claim 15, comprising an amino acid sequence of SEQ ID NO:1, orconserved variants thereof.
 18. The prokaryotic cation channel proteinof claim 11, wherein the functional eukaryotic cation channel proteincomprises a eukaryotic potassium channel protein, a eukaryotic sodiumchannel protein, or a eukaryotic calcium channel protein.
 19. Theprokaryotic cation channel protein of claim 11, wherein said functionaleukaryotic cation channel protein is endogenously produced in aeukaryotic organism comprising insects or mammals.
 20. The prokaryoticcation channel protein of claim 19, wherein said eukaryotic organismcomprises Drosophila melanogaster, Homo sapiens, C. elegans, Musmusculus, Arabidopsis thaliana, paramecium tetraaurelia or Rattusnovegicus.
 21. The prokaryotic cation channel protein of claim 11,mutated to mimic a eukaryotic cation channel protein comprising an aminoacid sequence comprising SEQ ID Nos: 4, 5, 6, 8, 9, 10, 11, 12, 13, or14.
 22. The prokaryotic cation channel protein of claim 21, wherein saidprokaryotic channel protein is a potassium channel protein fromStreptomyces lividans comprising an amino acid sequence of SEQ ID NO:1,said eukaryotic cation channel is a potassium channel protein comprisingan amino acid sequence of SEQ ID NO:4, and said mutated prokaryoticchannel protein comprises an amino acid sequence of SEQ ID NO:16, orconserved variants thereof.
 23. The prokaryotic cation channel proteinof claim 22, wherein said mutated porkaryotic channel protein is encodedby an isolated nucleic acid molecule comprising a DNA sequence of SEQ IDNO:17, or degenerate variants thereof.
 24. An isolated nucleic acidmolecule which encodes a mutant K⁺ channel protein, comprising a DNAsequence of SEQ ID NO:17, or degenerate variants thereof.
 25. Anisolated nucleic acid molecule hybridizable to the isolated nucleic acidmolecule of claim 24 under standard hybridization conditions.
 26. Theisolated nucleic acid molecule of claim 24, detectably labeled.
 27. Theisolated nucleic acid molecule of claim 25, detectably labeled.
 28. Thedetectably labeled isolated nucleic acid molecule of either of claims 26or 27, wherein said detectable label comprises radioactive isotopes,compounds which fluoresce, or enzymes.
 29. The isolated nucleic acidmolecule of either of claims 24 or 25, which encode a polypeptidecomprising an amino acid sequence of SEQ ID NO:16, or conserved variantsthereof.
 30. An isolated polypeptide comprising an amino acid sequenceof SEQ ID NO:16, or conserved variants thereof.
 31. An antibody having apolypeptide of claim 30 as an immunogen.
 32. The antibody of claim 31,wherein said antibody is a monoclonal antibody.
 33. The antibody ofclaim 32, wherein said antibody is a polyclonal antibody.
 34. Theantibody of claim 33, wherein said antibody is a chimeric antibody. 35.The antibody of any of claims 31-34 detectably labeled.
 36. The antibodyof claim 35, wherein said detectable label comprises an enzyme, achemical which fluoresces, or a radioactive isotope.
 37. A cloningvector comprising an isolated nucleic acid residue of either of claims24 or 25, and an origin of replication.
 38. The cloning vector of claim37, wherein said cloning vector is selected from the group consisting ofE. coli, bacteriophages, plasmids, and pUC plasmid derivatives.
 39. Thecloning vector of claim 37, wherein bacteriophages further compriselambda derivatives, plasmids further comprise pBR322 derivatives, andpUC plasmid derivatives further comprise pGEX vectors, or pmal-c, pFLAG.40. An expression vector comprising an isolated nucleic acid molecule ofeither of claims 24 or 25, operatively associated with a promoter. 41.The expression vector of claim 40, wherein said promoter is selectedfrom the group consisting of the immediate early promoters of hCMV,early promoters of SV40, early promoters of adenovirus, early promotersof vaccinia, early promoters of polyoma, late promoters of SV40, latepromoters of adenovirus, late promoters of vaccinia, late promoters ofpolyoma, the lac the trp system, the TAC system, the TRC system, themajor operator and promoter regions of phage lambda, control regions offd coat protein, 3-phosphoglycerate kinase promoter, acid phosphatasepromoter, and promoters of yeast α mating factor.
 42. A unicellular hosttransformed with an expression vector of claim
 40. 43. The unicellularhost of claim 42, wherein said host is selected from the groupconsisting of E. coli, Pseudonomas, Bacillus, Strepomyces, yeast, CHO.R1.1, B-W, L-M, COS1, COS7, BSC1,BSC40, BMT10 and St9 cells.
 44. Amethod of producing a mutant cation channel protein comprising an aminoacid sequence of SEQ ID NO:16, or conserved variants thereof, comprisingthe steps of: a) culturing a unicellular host of claim 42 underconditions that provide for expression of said mutant cation channelprotein; and b) recovering said mutant cation channel protein from saidunicellular host.
 45. A method of screening for compounds whichselectively bind to a potassium ion channel protein comprising: (a)complexing a functional two-transmembrane-domain-type potassium ionchannel protein to a solid, support; (b) ontacting the complexedprotein/solid support with an aqueous solution said solution containinga compound that is being screened for the ability to selectively bind tothe ion channel protein; (c) determining whether the compoundselectively binds to the ion channel protein with the provisoes that thepotassium ion channel protein is in the form of a tetrameric protein;and, when the protein is mutated to correspond to the agitoxin2 dockingsite of a Shaker K⁺ channel protein by substituting amino acid residuespermitting the mutated protein to bind agitoxin2, the protein will bindagitoxin 2 while bound to the solid support, said substituting ofresidues being within the 36 amino acid domain defined by −25 to +5 ofthe selectivity filter where the 0 residue is either the phenylalanineor the tyrosine of the filter's signature sequence selected from thegroup consisting of glycine-phenylalanine-glycine orglycine-tyrosine-glycine.
 46. A method of claim 45 wherein the solidsupports are selected from the group comprising: cobalt, insolublepolystyrene beads, PVDF, and polyethylene, glycol.
 47. A method of claim45 wherein the two-transmembrane-domain-type ion channel protein is aprokaryote.
 48. A method of claim 45, wherein thetwo-transmembrane-domain-type ion channel protein is from Steptomyceslividans.
 49. A method of claim 45 wherein thetwo-transmembrane-domain-type ion channel protein is KcsA.
 50. A methodof claim 45 wherein the two-transmembrane-domain-type ion channelprotein is mutated from a wild-type protein.
 51. A method of claim 50where the mutation is within the 36 amino acid domain defined by −25 to+5 of the selectivity filter where the 0 residue is either thephenylalanine or the tyrosine of the filter's signature sequenceselected from the group consisting of glycine-phenylalanine-glycine orglycine-tyrosine-glycine.
 52. A method of claim 50 wherein the mutationdeletes a subsequence of the native amino acid sequence and replacesthat the native with a subseqeunce from the corresponding domain of asecond and different ion channel protein.
 53. A method of claim 52wherein the second ion channel protein is from a eukaryote.
 54. A methodclaim 45 wherein the aqueous solution comprises a non-ionic detergent.55. A non-natural and functional two-transmembrane-domain-type potassiumion channel protein wherein the non-natural protein is mutated in itsamino acid sequence from a corresponding natural protein whereby themutation does not prevent the non-natural protein from binding agitoxin2when the non-natural protein is further mutated to correspond to theagitoxin2 docking site of a Shaker K⁺ channel protein said docking sitecreated by substituting amino acid residues selected from within the 36amino acid domain defined by −25 to +5 of the Shaker K⁻ selectivityfilter where the 0 residue is either the phenylalanine or the tyrosineof the filter's signature sequence selected from the group consisting ofglycine-phenylalanine-glycine or glycine-tyrosine-glycine.
 56. Anon-natural protein of claim 55 wherein the protein binds to a channelblocking protein toxin with at least a 10 fold increase in affinity overthe native ion channel.
 57. A non-natural protein of claim 55 whereinthe natural protein is the KcsA from Streptomyces lividans.
 58. A methodof assessing the adequacy of the structural conformation of atwo-transmembrane-domain-type potassium ion channel protein for highthrough put assays comprising the steps of: (a) complexing atwo-transmembrane-domain-type potassium ion channel protein having atetrameric form to a non-lipid solid support under aqueous conditions;(b) contacting the complexed two-transmembrane-domain-type potassium ionchannel protein with a substance known to bind to thetwo-transmembrane-domain-type potassium ion channel protein when boundto lipid membrane wherein the substance also modulates potassium ionflow in that channel protein; and, (c) detecting the binding of thesubstance to the complexed two-transmembrane-domain-type potassium ionchannel protein.
 59. A method of claim 58 wherein thetwo-transmembrane-domain-type potassium ion channel protein is mutatedfrom a wild type two-transmembrane-domain-type potassium ion channelprotein by substitution of amino acids.
 60. A method of claim 58 whereinthe contacting is done in the presence of a non-ionic detergent.
 60. Amethod of claim 58 where in the substance is a channel blocker.
 62. Amethod of claim 58 wherein the substance is a toxin.
 63. A prescreeningmethod for identifying potential modulators of potassium ion channelfunction comprising: (a) binding a soluble potassium ion channel proteinto a solid support where the ion channel has the scaffold of atwo-transmembrane-domain-type potassium ion channel and has a tetramericconfirmation; (b) contacting the soluble potassium ion channel proteinof step i with a compound in an aqueous solution; and, (c) determiningthe binding of the compound to the soluble potassium ion channelprotein.
 64. A method of claim 63 wherein the contacting takes place inthe presence of a detergent.
 65. A method of claim 63 wherein the ionchannel can pass potassium ions when expressed in a cell.
 66. A methodof claim 63 which further comprises the contacting of the compound tocell expressing a two-transmembrane-domain-type potassium ion channelprotein said cell cultured in an aqueous media containing potassium anddetermining modulation of potassium flow between the inside of the celland the media.
 67. A column comprising a solid support having boundthereto an ion channel having the scaffold of atwo-transmembrane-domain-type potassium ion channel and having atetrameric confirmation.
 68. A column of claim 25 wherein the ionchannel is a non-natural and functional two-transmembrane-domain-typepotassium ion channel protein wherein the non-natural protein is mutatedin its amino acid sequence from a corresponding natural protein wherebythe mutation does not prevent the non-natural protein from bindingagiroxin2 when the non-natural protein is further mutated to correspondto the agitoxin2 docking site of a Shaker K⁺ channel protein saiddocking site created by substituting amino acid residues selected fromwithin the 36 amino acid domain defined by −25 to +5 of the Shaker K⁺selectivity filter where the 0 residue is either the phenylalanine orthe tyrosine of the filter's signature sequence selected from the groupconsisting of glycine-phenylalanine-glycine or glycine-tyrosine-glycine.